Methods Of HLA Engineering and Treatments For Autoimmunity

ABSTRACT

Methods of preventing or treating autoimmune disease are disclosed. In some cases, subjects with having or at risk of developing autoimmune disease are identified as possessing one or more autoimmunity-susceptibility HLA alleles at one or more HLA loci. In many cases, the HLA loci are selected from Class I and Class II loci, for example Class I A, B, and C, and Class II DQ, DR, and DP. In many cases, subjects suffering from or at risk of developing an autoimmune disease may be administered a plurality engineered autologous HSCs modified to carry and express a variant susceptibility allele having at least one mutation in the antigen binding cleft that alters antigen binding and/or specificity of that variant HLA molecule. In many embodiments, the engineered HSCs are CD34+ immune cells that express one or more modified HLA proteins.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims benefit of and priority pursuant to 35 U.S.C. §119(e) of U.S. provisional patent application No. 63/186,770 entitled“HLA Engineering Methods, Compounds, and Compositions for Treatment ofAutoimmunity,” filed on 10 May 2021, which is also hereby incorporatedby reference in its entirety. This application is concurrently filedwith related PCT applications entitled “Methods of HLA Engineering andTreatments for Autoimmunity”, “Engineered HLA Alleles for TreatingAutoimmunity”, and “Pocket Engineering of HLA Alleles for TreatingAutoimmunity.”

FIELD

The disclosed compositions, methods, and systems are directed totreatment and prevention of autoimmune conditions.

Sequence Listing

The instant application contains a Sequence Listing which has beensubmitted electronically in XML format and is hereby incorporated byreference in its entirety. Said XML copy, created on Jul. 18, 2022, isnamed P292109US02.xml and is 155 kilobytes in size. The Sequence Listingdoes not extend beyond the scope of the specification, and does not addnew matter.

BACKGROUND

Autoimmunity refers to pathologic conditions in which the body's immunesystem mistakenly identifies healthy tissues and cells as foreign andattacks them. Any disease that results from this mistaken immuneresponse is termed an autoimmune disease, disorder or condition. Whilesome autoimmune diseases such as rheumatoid arthritis (RA), Type 1diabetes (T1 D), and multiple sclerosis (MS) are more prevalent thanothers, collectively they pose a serious public health challengeimpacting millions of people across the world. Commonly, patients withan autoimmune disease suffer from various symptoms that, withoutlimitation, can range from mild including fatigue, fever, muscle aches,joint pain and swelling, skin problems, abdominal pain, and digestionproblems, to more severe, which can include decreased mobility, loss ofvision, and organ failure.

Autoimmune disease can have various molecular, cellular, andphysiological bases. Generally, autoimmunity is the result of adysregulated immune system, which may stem from genetic or environmentalfactors, resulting in a subject's immune system turning on itself.Ideally, under normal circumstances, a healthy immune system recognizesand fights off foreign bodies (e.g., microbes, viruses, proteins, andnucleic acids). However, to do this effectively, it must be trained toavoid attacking the subject's own tissues, cells, proteins, and nucleicacids.

Human leukocyte antigen (HLA) refers to a group of related genes codingfor proteins involved in immune function. HLA Class I and II proteinsare cell-surface proteins with peptide clefts for presenting peptides toT-cell receptors. The HLA complex of genes reside on the Short Arm ofhuman Chromosome 6. Reference to alleles of the HLA proteins has awell-known nomenclature. For example, DRB1*01:01:01:01, as is well knownto the skilled HLA researcher, refers to an allele of the DRB1 gene ofthe HLA complex, the first two values after the HLA gene designation andseparated by the ‘*’ (in this example the ‘01:01’) refers to the allelegroup or level, and variations at the protein sequence level—forexample, DRB1*01:01 and DRB1*01:02 differ by two amino acids in thepeptide binding region. The 3rd field (here, the third ‘01’) indicates adifference in the genetic sequence that, due to degeneracy of thegenetic code, does not change the amino acids and are thereforeimmunologically identical—i.e., DRB1*01:02:01 is immunologicallyidentical to DRB1*01:02:02. The last field (i.e., the final ‘01’)indicates differences in the genetic sequence that occur outside of thecoding region of the protein (introns, promoters, etc.)— thus,DRB1*01:01:01:01 and a hypothetical DRB1*01:01:01:02 would be identicalat the immunological and genetic levels, within the coding sequence, buthave different non-coding sequences. This type of change typically mayaffect expression levels. Thus, by convention, as referenced herein theengineered HLA alleles are generally described using the first twofields.

HLA is the main genetic factor related to autoimmune diseases,accounting for approximately half of known genetic predisposition.Although more than 200 associations between HLA and disease have beendescribed, the underlying pathogenic mechanisms remain poorly defined.Initially, the particular genetic characteristics of HLA, and thecomplex interaction with other genes and environment have preventedfurther clinically meaningful developments in this field. There is agreater need for dissecting and understanding the role of HLA in diseasesusceptibility.

One autoimmune disease, rheumatoid arthritis, or RA, is characterized byinflammation of the joint capsule synovia, resulting in an infiltrationof macrophages, neutrophils, T cells, and B cells. This culminates inextensive joint destruction, disability, and reduced quality of life.The persistent inflammation associated with RA also increases the riskof developing ischemic heart and respiratory disease, resulting in earlymortality. RA occurs in approximately 1% of the world population, withan estimated 1.3 million affected in the United States of America (US)alone. RA occurs more frequently in women over the age of 40 and inlong-term smokers. Billions of dollars of direct healthcare costs areassociated with the treatment of RA annually, and total annual societalcosts of RA (direct, indirect and intangible) are estimated to reachtens of billions of dollars in the US alone.

Treatment of RA requires a systematic approach with frequent monitoringof disease activity and medication side effects to determine the optimaltherapeutic regimen appropriate for the patient. There are currently adiverse range of approved therapeutic agents to control symptoms, managepain, and limit joint damage. Current medications for RA includenon-steroidal anti-inflammatory drugs (NSAIDs), analgesics,corticosteroids, synthetic disease-modifying antirheumatic drugs(DMARDs), and biologic agents. DMARD treatments globally target majorcomponents of the immune system to halt progression of RA and requiresustained administration to maintain remission. This puts patients atrisk of developing unwanted side effects, serious infections,malignancy, and organ toxicity; patients can also develop anti-drugantibodies (ADAs) against biologics that neutralize their effects.Furthermore, between approximately 6% and 21% of patients fail toachieve sufficient response to adequately manage disease with currenttreatments. Such patients are commonly referred to as refractory RApatients.

Existing treatments for autoimmune diseases target the symptoms and notthe root cause of the diseases. Many autoimmune diseases, like RA, areinitiated by the presentation of modified self-peptides by a subset ofHLA alleles.

Transplantation of hematopoietic stem cells (HSCs) to cure RA has beenunsuccessful at safely conferring long-term remission. First, autologoustransplants employ a short course of chemotherapy to reset the immunesystem and are relatively safe but, rather than address the rootproblem, they simply re-populate the bone marrow with the same,problematic cells that allowed RA to develop in the first place.Secondly, allogeneic bone marrow transplants from HLA-matched donorsalso exhibit a high rate of relapse due to the fact that the same HLAalleles were used to replace the patient's bone marrow. Moreover, thistechnique is associated with graft-versus-host disease (GVHD), making itan unacceptable therapeutic strategy. A recent meta-analysis of 17studies involving 155 unique patients with RA who had undergoneautologous HSC transplants demonstrated that remission was notmaintained beyond 2 years.

The National Institutes of Health (NIH), in 2005, reported that as manyas 23.5 million people in the U.S. may suffer from autoimmune diseases,which, in most cases, lack cures. The lack of cures results in manypatients suffering from debilitating symptoms, loss of organ function,reduced productivity at work, and high medical expenses. What is neededare effective therapies to treat autoimmune diseases.

Herein, Applicants describe techniques that target the HLA alleleassociated with autoimmune diseases and use this information to createtailored treatments comprising one or more autologous HSCs wherein thetarget HLA allele has been engineered to have altered antigen bindingaffinity and/or specificity.

SUMMARY

Herein, to address the foregoing and other shortcomings in existingtreatment and management of autoimmune disease, Applicants havedeveloped methods that identify and target HLA alleles associated withthe disease and use this information to create tailored treatmentsinvolving one or more autologous HSCs wherein the target HLA allele hasbeen engineered to have altered self-antigen binding affinity and/orspecificity.

Disclosed herein are methods and compositions useful in reducingautoimmunity in a subject suffering from or at risk of developing anautoimmune disease, disorder, or condition. Such diseases, disorders,and conditions include, without limitation, rheumatoid arthritis (RA),celiac disease, diabetes mellitus type 1, systemic lupus erythematosus(SLE), multiple sclerosis (MS), myelin oligodendrocyte glycoproteinantibody disorders (MOGAD), myasthenic syndromes and neuromyelitisoptica (NMO), ankylosing spondylitis, Behget's syndrome, Birdshotuveitis, narcolepsy, narcolepsy type 1 (NT1; previously termednarcolepsy with cataplexy), Kawasaki disease, Crohn's disease,psoriasis, dermatomyositis (DM), Addison's disease, irritable-bowlsyndrome (IBS), Graves' disease, Henoch-Schönlein purpura (HSP),sarcoidosis, Sjögren's syndrome, eosinophilic granulomatosis withpolyangiitis, Hashimoto's disease, idiopathic thrombocytopenic purpura,polymyositis (PM), paraneoplastic neurological syndromes (PNS),autoimmune encephalitis, lupus nephritis (LN), myasthenia gravis (MG),psoriatic arthritis, graft rejection, graft-versus-host disease (GVHD),an unwanted delayed-type hypersensitivity reaction, T-cell mediatedpulmonary disease, neuritis, vitiligo, autoimmune pancreatitis,inflammatory bowel diseases, ulcerative colitis, glomerulonephritis,scleroderma, autoimmune thyroid diseases, asthma, autoimmuneuveoretinitis, pemphigus vulgaris, pulmonary fibrosis or idiopathicpulmonary fibrosis, primary biliary cirrhosis, and pernicious anemia.Various autoimmune diseases are associated with the presence of one ormore alleles of the human leukocyte antigen (HLA) genes, which is agroup of related genes coding for proteins involved in immune function.HLA Class I and Class II proteins are cell-surface proteins with peptideclefts for presenting peptides to T-cell receptors. The HLA complex ofgenes reside on the short arm of human Chromosome 6.

In one aspect, methods of modifying an HLA allele associated with anautoimmune disease are provided. One such method includes the steps ofidentifying autoimmunity-susceptibility HLA alleles; identifying targetamino acid positions within a binding cleft of a susceptibility HLAallele-coded protein, wherein the target amino acid position has anidentity that is different in an auto-immunity-resistant HLA allele;modifying the amino acid identity of the target amino acid position tothe identity of the same amino acid position in theauto-immunity-resistant HLA allele to create a modifiedautoimmunity-susceptibility HLA allele, wherein a protein coded by themodified autoimmunity-susceptibility HLA allele possesses alteredbinding affinity for at least one self-peptide.

In a related aspect of the present disclosure, methods of treating asubject suffering from or at risk of developing an autoimmune diseaseare provided. One such method includes the steps of identifying anautoimmunity-susceptibility HLA allele within an HLA complex of thesubject; isolating a plurality of CD34+ immune cells from the subject;and modifying the CD34+ immune cells to create modified CD34+ immunecells expressing a modified autoimmunity-susceptibility HLA allele. Themodified autoimmunity-susceptibility HLA allele encodes a protein withaltered binding affinity for at least one self-peptide as compared to aprotein coded for by the autoimmunity-susceptibility HLA allele.

In certain embodiments according to the present disclosure, methods areprovided for identifying autoimmune conditions related to antigenpresentation by HLA Class I and Class II proteins that are treatablewith engineered autologous-HLA expressing hematopoietic cells. In manyembodiments, the HLA loci are selected from Class I A, B, and C, andClass II DP, DR, and DQ. In some embodiments, HLA genes, alleles, andproteins may include one or more of HLA-A*02, HLA-A*03, HLA-A*29,HLA-B*07, HLA-B*08, HLA-B*27, B*27:03 B*27:05, B*27:09, HLA-B*51,HLA-B*54, HLA-B*57, HLA-C*06, HLA-C*18, HLA-DPA1*02, HLA-DPB1*13,HLA-DQA1*02, HLA-DQA1*03, HLA-DQA1*05, HLA-DQB1*02, HLA-DQB1*03,HLA-DQB1*06, HLA-DRB1*01, HLA-DRB1*04, HLA-DRB1*07, HLA-DRB1*08,HLA-DRB1*11, HLA-DRB1*15, HLA-DRB1*16, and variants of those HLAs. Thedisclosed methods include, in certain embodiments, the steps ofidentifying one or more HLA alleles associated with susceptibility(susceptibility allele) to a specific autoimmune disease and one or morealleles of the same HLA gene that are associated with resistance(resistance allele) to the specific autoimmune disease, identifying oneor more variable amino acid positions within the antigen binding grooveof the HLA gene, wherein the variable amino acid position of thesusceptible allele has a first identity and the variable amino acidposition of the resistance allele has a second identity.

Certain embodiments of the present disclosure are premised in part onthe discovery of causal associations between specific autoimmunediseases and specific HLA alleles. For example, certain embodiments arebased on the association of Type 1 diabetes with DQB1*02 and/or DQB1*03,and in particular with DQB1*02:01 and/or DQB1*03:02. In someembodiments, rheumatoid arthritis is associated with DRB1*04 andDRB1*01, in particular DRB1*04:01, DRB1*04:05, and DRB1*01:01. In somesuch embodiments, multiple sclerosis is associated with DRB1*15, inparticular DRB1*15:01. In some such embodiments, celiac disease isassociated with DQB1*02, in particular DQB1*02:01. In some suchembodiments, NMO is associated with DRB1*03, in particular DRB1*03:01.In some such embodiments, Behget's syndrome is associated with B*51 orB51. In some cases, psoriasis may be associated with C*06, B*57,DRB1*07, and/or DQB1*03. In some cases, Birdshot uveitis may beassociated with A*29. In some cases, narcolepsy may be associated withDQB1*06, in particular DQB1*06:02. In some cases, myasthenia gravis maybe associated with A*03, B*07, DR2 (DRB1*15 and/or DRB1*16) and and/orDR4 (DRB1*04). In some cases, Kawasaki disease may be associated withB*54, in particular amino acid positions 91, 104, and 329. In somecases, inflammatory bowel disease may be associated with DRB1*01, inparticular DRB1*01:03. In some cases, systemic sclerosis may beassociated with DRB1*11, DPB1*13, B*08, DQA1*02:01, DQA1*05, DRB1*08,DRB1*07, DPA1*02, DQB1*03, in particular DRB1*11:04, DPB1*13:01,B*08:01, DQA1*02:01, DQA1*05:01, DRB1*08:01, DRB1*07:01, DPA1*02:01,DQB1*03:01.

Also disclosed herein are compounds and compositions useful in treatingor preventing autoimmune conditions. In many embodiments, the disclosedcompounds and compositions include one or more engineered immune cellcomprising a modified HLA allele. In most embodiments, the modified HLAallele is an edited protein molecule and contains at least one aminoacid mutation within the peptide binding cleft of the HLA protein codedfor by the modified HLA allele. In other embodiments, the modified HLAallele is an edited nucleic acid molecule coding for an edited HLAprotein, wherein the edited nucleic acid contains at least one codoncoding for an amino acid mutation within the peptide binding cleft ofthe edited HLA protein. In most embodiments, the amino acid mutation isnot at the T-cell receptor interface. In many embodiments, the modifiedHLA allele may be carried, contained in, or expressed by an engineeredimmune cell. In many embodiments, the engineered immune cells areautologous cells—i.e., they are obtained from the subject being treatedfor the autoimmune disease. In many embodiments, the engineered immunecell may be comprised within a composition, for example a therapeuticcomposition that may be administered to a subject suffering from or atrisk for an autoimmune disease. In many embodiments, the engineeredimmune cell may be an HSC.

Further disclosed are methods of making the disclosed compounds andcompositions. In many embodiments the methods comprise identifying oneor more HLA genes associated with an elevated incidence of a specificautoimmune disease, identifying one or more alleles of the HLA geneassociated with susceptibility (susceptibility allele(s)) and/or one ormore alleles associated with resistance (resistance allele(s)) to thespecific autoimmune disease, identifying one or more variable amino acidpositions within the antigen binding groove of the HLA molecule, whereinthe variable amino acid position of the susceptible allele has a firstidentity and the variable amino acid position of the resistant allelehas a second identity. In certain embodiments, the methods of making thedisclosed compounds further comprise creating an engineered HLA moleculeof the susceptible allele wherein the identity of the amino acid at thevariable position is the second identity. In some embodiments, theengineered HLA molecule is coded for by an expression vector or anengineered genomic sequence.

Also disclosed are various methods of treating subjects in need with thedisclosed therapies, wherein treatment comprises administration of oneor more engineered antigen presenting cells having at least one mutatedamino acid within an MHC antigen binding region (e.g., an antigenbinding groove of an HLA protein). In many embodiments, the treatmentmethods include isolating one or more cells from a donor. In manyembodiments, the isolated cell is a HSC. In many embodiments, the methodfurther comprises the step of modifying the HSC to create an engineeredHSC. The engineered HSC comprises an engineered HLA allele (edited HLAallele, variant HLA allele, modified HLA allele) having altered bindingspecificity or affinity for a self-antigen or a variant self-antigen. Insome embodiments, the modified HSC comprises one or more of a nucleicacid sequence coding for the engineered HLA allele in its genomicsequence or one or more expression vectors comprising a nucleic acidsequence coding for the engineered HLA allele. In many embodiments, themodified HSC may engraft in the subject's bone marrow and produce one ormore modified antigen presenting cells.

Disclosed herein are various compositions for treating a subject at riskof developing or suffering from an autoimmune disease. Inrepresentative, specific embodiments, the compositions comprise a DNAsequence selected from the group consisting of SEQ ID NO:59-96.

In some certain embodiments according to the present disclosure,susceptibility to the autoimmune disease is associated with an HLA-DRB1gene, for example DRB1*01, DRB1*03, DRB1*04, DRB1*07, DRB1*09, DRB1*10,DRB1*11, DRB1*12, DRB1*13, DRB1*14, DRB1*15, and DRB1*16. In manyembodiments the autoimmune disease is associated with an allele ofHLA-DRB1 selected from DRB1*01:01, DRB1*01:02, DRB1*01:03, DRB1*03:01,DRB1*04:01, DRB1*04:02, DRB1*04:03, DRB1*04:04, DRB1*04:05, DRB1*04:08,DRB1*07:01, DRB1*09:01, DRB1*10:01, DRB1*11:01, DRB1*11:02, DRB1*11:03,DRB1*12:01, DRB1*13:01, DRB1*14:01, DRB1*15:01, DRB1*15:02, andDRB1*16:01. In related embodiments, the compositions comprise aDRB1*01:01 protein or DNA coding region therefor comprising a mutationat position selected from L67, Q70, V85, G86, R71 (positions of aminoacids are in reference to the mature protein sequence as presented atebi.ac.uk/ipd/imgt/hla), and combinations thereof, for example, withoutlimitation, L671, Q70D, V85A, G86V, R71E, and combinations thereof. Forexample, in some embodiments, the composition comprises a variantDRB1*03:01 protein or coding region comprising a mutation at positionV86, for example V86L or V86M. In some embodiments, the compositioncomprises a variant DRB1*04:03, DRB1*04:04 DRB1*04:05, and DRB1*04:08protein or coding region comprising a mutation at position R71, forexample R71E. In some embodiments, the composition comprises a variantDRB1*13:01 protein or coding region comprising a mutation at positionV86, for example V86L or V86M. In some embodiments, the compositioncomprises a variant DRB1*15:01 protein or coding region comprising amutation at position F47, A71, or V86, for example F47Y, A71R, V86L,V86M, and combinations thereof.

In some embodiments of the present disclosure, susceptibility to theautoimmune disease is associated with an HLA-DRB3, HLA-DRB4, or HLA-DRB5gene, for example HLA-DRB3*01, HLA-DRB3*02, HLA-DRB3*03, DRB4*01, andDRB5*01. For example, in certain embodiments, the autoimmune disease isassociated with an allele of HLA-DRB3/4/5 selected from DRB3*01:01,DRB3*02:02, DRB3*03:01, DRB4*01:01, DRB4*01:03, and DRB5*01:01.

In additional embodiments according to the present disclosure,susceptibility to the autoimmune disease is associated with the HLA-DQAand/or HLA DQB genes. For example, in certain embodiments, theautoimmune disease is associated with an allele of HLA-DQA1 and/orselected from DQA1*01, DQA1*03, DQA1*05, DQB1*02, DQA1*03, DQB1*05,DQB1*06, and combinations thereof, for example DQ5, DQA1*01:01 andDQB1*05:01; DQ6, DQA1*01:02 and DQB1*06:02; DQ2, DQA1*05:01 andDQB1*02:01; DQ2 Trans, DQA1*03:01 & DQB1*02:01; DQ8, DQA1*03:01 andDQB1*03:02; DQ8 trans, DQA1*05:01 and DQB1*03:02; DQA1*05:05 andDQB1*03:01; and DQA1*03:01 and DQB1*03:01. In some such embodiments, atleast one modified or variant HLA is engineered having at least onesubstitution within the antigen binding groove, for example position 57or 71, wherein the mutation is A57D, K71E, K71T, or combinationsthereof.

In further embodiments according to the disclosure, susceptibility tothe autoimmune disease is associated with the HLA-B gene. For example,in some embodiments, the autoimmune disease is associated with an alleleof B27 and/or selected from B*27:03 B*27:05, and B*27:09. In manyembodiments, the mutation may be at a position selected from anypolymorphic position within the antigen binding groove, for exampleposition 59 or 116, wherein the mutation is Y59H, D116H, or combinationsthereof.

Further disclosed herein are methods for identifying positions of HLAalleles that, when mutated, may be useful in reducing or eliminatingsusceptibility to, or symptoms of, autoimmunity. The methods include, inmany embodiments, comparing a cohort of individuals suffering from anautoimmune disease, identifying specific HLA gene allele(s) associatedwith disease susceptibility (susceptibility allele), identifyingspecific HLA gene allele(s) associated with disease resistance(resistance allele), identifying polymorphic amino acid positions,located within the antigen binding groove of the HLA molecule, betweenthe resistant allele (or resistance allele) and the susceptibilityallele (or susceptible allele)—that is, positions where the amino acididentity in the resistant allele is different than the identity in thesusceptibility allele. As one example, residues of the DRB1 gene locatedwithin the antigen binding groove include 8-14, 16, 25-26, 28, 30-33,37-38, 40, 47, 57-60, 67, 70-71, 73-74, 77-78, 85-86, and 93. In relatedembodiments, the methods further comprise engineering the susceptibilityallele to include the resistant allele's amino acid identity at thepolymorphic position. In certain embodiments according to the presentdisclosure, expressing such engineered HLA molecule or molecules on oneor more antigen presenting cells (APCs) prevents, treats, or amelioratesautoimmune disease in a subject.

Also disclosed herein are engineered HLA molecules having alteredantigen binding and/or specificity compared to a non-engineered HLAmolecule. In many embodiments, the antigen may be selected from variouspeptides including modified peptides, citrullinated peptides, hybridpeptides, nucleic acids, etc. In some embodiments, the hybrid peptide isa hybrid insulin peptide. In some embodiments, the peptide is selectedfrom ENPVVHFFKNIVTPRTPPP SEQ ID NO: 123, LVRYWISAFP SEQ ID NO: 122,FFRDHSYQEEA SEQ ID NO: 121, AQGTLSKIFKLGGRDSRSGSPMARR SEQ ID NO: 124,GQVELGGWSKMDQLA SEQ ID NO: 97, GQVELGGGNAVEVLK SEQ ID NO: 98,GQVELGGGSSPETLI SEQ ID NO: 99, SLQPLALEAEDLQV SEQ ID NO: 100,HLVEELYLVAGEEG SEQ ID NO: 102, AMMIARFKMFPEVKEKG SEQ ID NO: 101,SHLVEALYLVCGERG SEQ ID NO: 104, RSQVETDDLILKPGV SEQ ID NO: 105,SQVETDDLILKPGVV SEQ ID NO: 106, PGIAGFKGEQGPKGE SEQ ID NO: 107,IFDSRGNPTVEVDLF SEQ ID NO: 108, IFDS{CIT}GNPTVEVDLF SEQ ID NO: 109,SAVRLRSSVPGVR SEQ ID NO: 110, SAVRL{CIT}SSVPGVR SEQ ID NO: 111,QDFTNRINKLKNS SEQ ID NO: 112, QDFTN{CIT}INKLKNS SEQ ID NO: 113,ATEGRVRVNSAYQDK SEQ ID NO: 114, ATEG{CIT}VRVNSAYQDK SEQ ID NO: 115,ATIKAEFVRAETPYM SEQ ID NO: 116, ATIKAEFV{CIT}AETPYM SEQ ID NO: 117,AVRLQGSVAGVR SEQ ID NO: 118, PYHFKYHEKHFANAI SEQ ID NO: 119,PVSKMRMATPLLMQA SEQ ID NO: 120, PKYVKQNTLKLAT SEQ ID NO: 103, andcombinations thereof, wherein {CIT} indicates a deaminated arginineresidue, which may be referred to as a citrullinated residue.

Also disclosed are methods of occluding a pocket in a binding cleft ofan HLA allele, the methods comprising the steps of identifyingsusceptible HLA alleles, and identifying target amino acid position ator near a pocket of the antigen binding cleft, wherein the pocketdefines a recess at the bottom of the antigen binding cleft. The methodsmay further comprise substituting an amino acid having a side chainlarger than the target amino acid to create an occlusion HLA allele,wherein the side chain of the second amino acid extends into the recessat the bottom of the antigen binding cleft, and thereby occluding thepocket of the HLA allele. In various embodiments, the HLA allele may beselected from HLA-DRB1, HLA-DRB3, HLA-DRB4, and HLA-DRB5, and the pocketmay be pocket 1. In these embodiments, the target amino acid may be, forexample, position 86, and the identity of the substituted amino acid maybe selected from valine, methionine, and leucine. In many embodimentsthe HLA protein with the occlusion in the pocket may possess lowerbinding affinity for at least one self-peptide associated with anautoimmune disease, optionally wherein the at least one self-peptide isdeaminated, and further optionally wherein the target amino acid is notat the T-cell receptor binding interface.

Throughout the present disclosure, various publications may bereferenced. The disclosures of these publications in their entiretiesare hereby incorporated by reference into this application, to theextent allowed by law.

The present disclosure is sufficient to enable one skilled in the art topractice the present disclosure. The present disclosure is not to belimited in scope by the constructs described, because the describedembodiments are intended as illustrations of certain aspects of thepresent disclosure and any constructs that are functionally equivalentare within the scope of this disclosure.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

Embodiments will be readily understood by the following detaileddescription in conjunction with the accompanying drawings. Embodimentsare illustrated by way of example and not by way of limitation in theaccompanying drawings.

FIG. 1 is a schematic depiction of various pathways, interactions, andpharmaceutical interventions in autoimmunity.

FIG. 2 depicts a schematic depiction of a mouse TCR/CD4 interaction withan engineered humanized HLA-DR4/I-E^(d), and at bottom is a graph ofresults from studies on collagen sensitization as detected by ex vivoproliferation of CD4+ T cells, where symbols indicate samples fromindividual humanized DRB1*04:01, DRB1*01:01 and DRB1*04:01^(K71E) miceand bars indicate means. Data was analyzed by One-way ANOVA.

FIG. 3A shows a three-dimensional depiction of DRB1*04:01 identifyingposition K71 within the cleft and the antigen binding cleft occupied bya collagen peptide (left); the figure at the right shows the structureof DRB1*04:01^(K71E) and the absence of collagen peptide binding—acidicresidues are shaded in blue and basic residues are shaded in red.

FIG. 3B shows skin grafts in DRB1*04:01 recipients for representativemice on days 0 and 9 and all mice on days 15-18 are shown. Long-termengraftment is shown for DRB1*04:01^(K71E) transplants (day 70) in lowerright panels. Red scabbing indicates rejection of the graft.

FIG. 4 is a sequence alignment of DRB1*01:01, DRB1*11:01 and DRB1*15:01mature length proteins, according to embodiments of the disclosure.

FIG. 5 depicts antigen binding studies of DRB1*01:01, *15:01 and *11:01alleles, numbers in upper left corner of boxes are binding ratios of thepeptide compared to cells that do not express any HLA Class II moleculeand therefore do not bind peptide (negative control).

FIG. 6 depicts binding of autoimmune demyelination-associated peptidesto DRB1*15:01 and 15:02 single and double mutant embodiments, numbers inupper left corner of boxes are binding ratios compared to negativecontrols (light gray).

FIG. 7 depicts binding autoimmune demyelination-associated peptides toDRB1*15:01 alleles, and the effects of edits at positions 71 and 86;numbers in upper left corner of boxes are binding ratios compared tonegative controls.

FIG. 8 is a sequence alignment of DRB1*03:01, DRB1*07:01, and DRB1*09:01mature length proteins, according to embodiments of the disclosure.

FIG. 9 depicts aquaporin 4 peptides 5 and 6 binding to DRB1*03:01 andDRB1*07:01.

FIG. 10 is a sequence alignment of *04:01 and *04:05 mature lengthproteins, according to embodiments of the disclosure.

FIG. 11 depicts binding of three peptides associated with rheumatoidarthritis to the DRB1 allele *04:05 allele and the effect of a R71Eedit, according to embodiments of the disclosure. The ratios in theupper right corner of the box refers to a comparison with the negativecontrol (collagen) or compared to native form of vimentin and α-enolase.

FIG. 12 depicts native vs. A57D binding of the HIP8-NPY peptide acrossmultiple concentrations, where closed circle is the native allele, opencircle is the A57D mutation: Panel A, DQ2; Panel B, DQ8; Panel C, DQ2Trans; and Panel D, DQ8 Trans.

FIG. 13 depicts native vs. A57D binding of the HIP11-C peptide acrossmultiple concentrations, where closed circle is the native allele, opencircle is the A57D mutation: Panel A, DQ2; Panel B, DQ8; Panel C, DQ2Trans; and Panel D, DQ8 Trans.

FIG. 14 depicts native vs. A57D binding of the Insulin Mimotope acrossmultiple concentrations, where closed circle is the native allele, opencircle is the A57D mutation: Panel A, DQ2; Panel B, DQ8; Panel C, DQ2Trans; and Panel D, DQ8 Trans.

FIG. 15 (top) shows that the DQ2 T2 cell lines stimulates the E2 T cellclone much better than the parent EBV line. Introducing the A57Dmutation results in less stimulation of the E2 T cell: stimulation of E2T cell with DQ2 and DQ2 A57D at 10 uM and 20 uM preload concentrationsof HIP11 peptide, solid circles are DQ2, open circles are DQ2 A57D, anddiamonds are patients EBV transformed B cell line (bottom) showsstimulation of E2 T cell with DQ2 Trans and DQ2 Trans A57D at 10 uM and20 uM preload concentrations of HIP11 peptide, where solid circles areDQ2 Trans, open circles are DQ2 Trans A57D, diamonds are patients EBVtransformed B cell line.

FIG. 16 depicts various HLA-DQ alleles binding hybrid insulin peptides,numbers in upper left corner of boxes are binding ratios.

FIG. 17 depicts various HLA-DQ alleles binding diabetogenic peptides,according to embodiments of the disclosure. Numbers in upper left cornerof boxes are binding ratios compared to negative controls.

FIG. 18 depicts binding of hybrid insulin peptides to DRB1*03:01, *04:01and *15:01, according to embodiments of the disclosure. Numbers in upperleft corner of boxes are binding ratios compared to negative controls.

FIG. 19 depicts binding of diabetogenic peptides and influenzahemagglutinin peptide to DRB1*03:01, *04:01 and *15:01, according toembodiments of the disclosure. Numbers in upper left corner of boxes arebinding ratios compared to negative controls.

FIG. 20 depicts binding of hybrid insulin peptides to DRB3, DRB4 andDRB5 alleles, according to embodiments of the disclosure. Numbers inupper left corner of boxes are binding ratios compared to negativecontrols. The ‘common’ serologic names of these alleles (e.g., HLA-DR52)are shown above the allele name.

FIG. 21 depicts binding of diabetogenic peptides to DRB3, DRB4 and DRB5alleles, according to embodiments of the disclosure. Numbers in upperleft corner of boxes are binding ratios compared to negative controls.

FIG. 22 is a list of various antigens used in the present studies toinvestigate binding by gene-edited HLA molecules, according toembodiments of the present disclosure.

FIG. 23A is a three-dimensional representation of a DRB1 structureshowing location of Pocket 1, according to embodiments of the presentdisclosure, and a two-dimensional representation of amino acids'chemistry.

FIG. 23B depicts antigen binding studies of DRB1*04:01 and editedalleles of the present disclosure with pocket 1 mutations G86L and G86M,according to embodiments of the disclosure. Numbers in upper left cornerof boxes are binding ratios compared to negative controls.

FIG. 24 depicts binding of hybrid insulin peptides to DRB1*04:01 and twoedited alleles of the present disclosure with pocket 1 mutations G86Land G86M, according to embodiments of the disclosure. Numbers in upperleft corner of the boxes are binding ratios compared to negativecontrols.

FIG. 25 depicts binding of neuroautoimmune peptides binding toDRB1*04:01 and edited alleles of the present disclosure with pocket 1mutations G86L and G86M, according to embodiments of the disclosure.Numbers in upper left corner of boxes are binding ratios compared tonegative controls.

FIG. 26 depicts binding of arthritogenic peptides to DRB1*04:01 andengineered alleles of the present disclosure with pocket 1 mutations,according to embodiments of the disclosure. Numbers in upper left cornerof boxes are binding ratios of citrullinated peptides compared to nativepeptides.

FIG. 27 depicts binding of native and citrullinated arthritogenicpeptides to DRB1*04:01 and engineered alleles of the present disclosurewith pocket 1 mutations, according to embodiments of the disclosure.Numbers in upper left corner of boxes are binding ratios compared to thenegative controls.

FIG. 28 is a list of representative HLA alleles, amino acid positions,and mutations according to embodiments of the present disclosure.

FIG. 29 lists mature protein sequences of various HLA alleles.

FIG. 30 lists cDNA sequences of various embodiments of susceptible andengineered HLA alleles.

DETAILED DESCRIPTION

Disclosed herein are various methods and compositions useful intreating, reducing, or eliminating autoimmune disease in a subjectsuffering from or at risk of developing the same. Such autoimmunediseases include, without limitation, rheumatoid arthritis (RA), celiacdisease, diabetes mellitus type 1, systemic lupus erythematosus (SLE),multiple sclerosis (MS), myelin oligodendrocyte glycoprotein antibodydisorders (MOGAD), myasthenic syndromes and neuromyelitis optica (NMO),ankylosing spondylitis, Behget's syndrome, Birdshot uveitis, narcolepsy,narcolepsy type 1 (NT1; previously termed narcolepsy with cataplexy),Kawasaki disease, Crohn's disease, psoriasis, dermatomyositis (DM),Addison's disease, irritable-bowl syndrome (IBS), Graves' disease,Henoch-Schönlein purpura (HSP), sarcoidosis, Sjögren's syndrome,eosinophilic granulomatosis with polyangiitis, Hashimoto's disease,idiopathic thrombocytopenic purpura, polymyositis (PM), paraneoplasticneurological syndromes (PNS), autoimmune encephalitis, lupus nephritis(LN), myasthenia gravis (MG), psoriatic arthritis, graft rejection,graft-versus-host disease (GVHD), an unwanted delayed-typehypersensitivity reaction, T-cell mediated pulmonary disease, neuritis,vitiligo, autoimmune pancreatitis, inflammatory bowel diseases,ulcerative colitis, glomerulonephritis, scleroderma, autoimmune thyroiddiseases, asthma, autoimmune uveoretinitis, pemphigus vulgaris,pulmonary fibrosis or idiopathic pulmonary fibrosis, primary biliarycirrhosis, and pernicious anemia.

In particular embodiments according to the present disclosure, thedisclosed autoimmune diseases are correlated with the presence of one ormore human leukocyte antigen (HLA) alleles. Applicants describe hereinmethods and compounds useful in ameliorating one or more symptoms ofautoimmune disease in a subject suffering therefrom. In manyembodiments, the methods may include identifying an autoimmunesusceptible HLA allele expressed by the subject's antigen presentingcells comparing the amino acid sequence of that susceptible HLA allelewith one or more HLA alleles associated with resistance to that sameautoimmune disease. In most embodiments, hematopoietic stem cells aremobilized and isolated from the subject, and the susceptible HLA alleleis modified or replaced with an engineered HLA allele comprising one ormore amino acid substitutions within the antigen binding cleft of theprotein coded for by the HLA allele, the specific identity of thesubstituted amino acid corresponds with the identity of that same aminoacid position in the HLA allele associated with resistance.

In certain embodiments, targeted engineering of the antigen presentingcleft of the HLA gene modifies binding specificity and/or affinity toone or more self-antigens. In most embodiments, a single amino acidwithin the cleft, that is hidden from TCR interrogation is mutated toalter peptide biding without directly affecting TCR binding. In mostembodiments, the disclosed HLA mutations result in HLA protein changesthat fail to trigger either rejection or GVHD in the patient. In mostembodiments, expression of the engineered HLA proteins on one or moreantigen presenting cells in a subject suffering an autoimmune diseasemay result in amelioratin of one or more symptoms associated with theautoimmune disease.

Also disclosed are methods of occluding a pocket in a binding cleft ofan HLA allele, the methods comprising the steps of identifyingsusceptible HLA alleles, and identifying target amino acid position ator near a pocket of the antigen binding cleft, wherein the pocketdefines a recess at the bottom of the antigen binding cleft. The methodsmay further comprise substituting an amino acid having a side chainlarger than the target amino acid to create an occlusion HLA allele,wherein the side chain of the second amino acid extends into the recessat the bottom of the antigen binding cleft, and thereby occluding thepocket of the HLA allele. In various embodiments, the HLA allele may beselected from HLA-DRB1, HLA-DRB3, HLA-DRB4, and HLA-DRB5, and the pocketmay be pocket 1. In these embodiments, the target amino acid may be, forexample, position 86, and the identity of the substituted amino acid maybe selected from valine, methionine, and leucine. In many embodimentsthe HLA protein with the occlusion in the pocket may possess lowerbinding affinity for at least one self-peptide associated with anautoimmune disease, optionally wherein the at least one self-peptide isdeaminated, and further optionally wherein the target amino acid is notat the T-cell receptor binding interface.

Applicant's present concept is presented in the diagram at FIG. 1 . TheHLA T cell receptor (TCR) interaction is central aspect in thepathogenesis of many autoimmune diseases, for example those describedabove. Current biologic agents targeting autoimmune disease tend totarget and block innate and adaptive pathways downstream from thissignal. This is problematic because these pathways engage in protectiveimmune responses against myriad pathogens and blocking them or alteringthem leaving the patient at risk for various opportunistic infections.

APCs are derived from HSC progenitors in the bone marrow. The presentlydescribed engineered HSCs replace the subjects APCs that presentantigens that lead to autoimmunity. The engineered HSCs express alteredHLA molecules, which will reduce, prevent, and/or resolve, prioractivation of autoreactive CD4+ T cells and their subsequent effects onchronic inflammatory cytokine production, macrophage activation, and Bcell autoantibody production.

Thus, in one aspect of the present disclosure, Applicants provide hereinthe ability to treat autoimmune diseases using engineered autologousHSCs comprising edited HLA proteins as presently described. Thedisclosed methods advantageously target specifically the underlyingetiology of the patient's autoimmune disease while avoiding broadeffects on other aspects of the patient's immune system.

Monocytes, macrophages, and dendritic cells (DCs) are the principal APCsthat help to initiate and maintain the disease state in many autoimmunediseases. For example, in RA, the cells maintain the hyperinflammatorystate in the joints associated with pain and debilitating progression ofjoint damage of the disease. However, these cells are short-lived andmust be replenished from CD34⁺HSCs in the bone marrow on a regularbasis. Monocytes, for example, typically survive in the blood for only afew days. However, if a monocyte migrates to an inflamed joint, they mayprogress to monocyte-derived DCs and macrophages and survive for weeksto months. Thus, Applicant's present disclosure describes replacing asubset of a patient's bone marrow with engineered HSCs that will producenew engineered monocytes, macrophages and DCs that no longer presentauto-immunogenic antigens, thus preventing activation of T-Cells and/orcausing autoreactive T cells to revert to a quiescent memory state.

The presently disclosed methods, compositions, and systems generally donot include depleting the patient's T cells and B cells prior toinfusion of engineered HSCs. Thus, the presently disclosed therapeuticmethods retain the patient's normal, innate, and adaptive immunity toinfection by microbial pathogens and recognition tumor antigens.

Selecting HLA Allele, Position, and Mutation for Expression byEngineered HSC

Disclosed herein are methods for selecting HLA alleles, target aminoacid positions within those alleles, and mutations at those positionsfor expression by engineered HSCs. In some embodiments, the disclosedmethods, compositions, and systems may include selecting and identifyingmore than one HLA allele, position, and/or mutation, modifying saidallele to create an engineered HLA allele with altered binding affinityfor at least one self-antigen as compared to an unmodified HLA allele.In many embodiments, the engineered HLA allele is expressed byengineered hematopoietic cells of a patient to be treated by thedisclosed therapy.

The disclosed methods may include identifying and/or selecting an HLAallele that is closely associated with increased risk of autoimmunity,which may be referred to as a susceptibility allele or susceptible HLAallele. In certain embodiments, the susceptible HLA allele is found ingreater than about 5% of patients with a specific autoimmune disease,for example greater than about 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%,14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%,28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, or moreand less than about 90%, 80%, 70%, 60%, 50%, 45%, 4%, 4%, 39%, 38%, 37%,36%, 35%, 34%, 33%, 32%, 31%, 30%, 29%, 28%, 27%, 26%, 25%, 24%, 23%,22%, 21%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%,7%, 6%, or 5%. In many embodiments, the susceptible HLA allele may befound in a smaller percentage of individuals that do not suffer from theidentified autoimmune disease (i.e., a control population or controls),for example, less than about 35%, 30%, 25%, 20%, 19%, 18%, 17%, 16%,15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, or 5%.

In the case of RA, DRB1*04:04, DRB1*01:01 or DRB1*04:01 may be selectedas an HLA allele for engineering. For example, although several DRB1alleles contain an arginine in position 71 and are at increased risk ofdeveloping RA, they are relatively uncommon. DRB1*01:01 is found at anallele frequency of 13% in RA patients, compared to 9.7% of controls andis the most common of these other ‘shared epitopes’. The allelesDRB1*04:03 (0.6%), *04:04 (9.1%), *04:05 (1.2%), *04:08 (1.7%) and*10:01 (2%) are all relatively rare among RA patients. In contrast,DRB1*04:01 is seen in 31% of RA patients (compared to 10% of controls,ρ=10⁻³). The frequency of DRB1*04:01 increases with the severity of thedisease to greater than 50% of refractory RA patients and 88% of themost severe form of RA (Felty's Syndrome). In addition, DRB1*04:01contributes the highest degree of susceptibility to RA.

The disclosed methods may include identifying and/or selecting a targetamino acid position within the susceptible HLA allele to be mutated tocreate the engineered HLA allele. In certain embodiments, the selectedtarget amino acid position (1) is not buried in HLA protein's structuralcore, (2) is positioned at or near the antigen binding cleft of the HLAprotein, (3) within the groove/cleft and not directly accessible to theT-cell receptor, i.e., is not at the TCR:HLA binding interface, and/or(4) alters binding affinity of at least one antigen to the engineeredHLA allele. In many embodiments, the disclosed target amino acidposition in a protein encoded by the selected susceptible HLA allelemolecule may have an identity that is different in another allele of thesame HLA gene that is not associated with susceptibility. Instead, thisother HLA allele may be associated with resistance to the sameautoimmune disease; this HLA allele may be referred to as a resistantHLA allele. For example, target amino acid position 71 in the matureprotein of RA-associated susceptible HLA allele DRB1*04:01 is lysine,while in the RA-associated resistant HLA protein, DRB1*04:02, position71 is glutamic acid.

According to certain embodiments, HLA engineering is optimized tominimize or avoid entirely the consequences of HLA mismatching. Amongrecipients of allogeneic bone marrow transplants, any HLA disparityincreases the risk of graft failure (rejection) and GVHD, so certainembodiments described herein include mutations within the antigenbinding groove/cleft of the HLA molecule. For example, the location ofK71 in DRB1*04:01 is below the upper surface (TCR interaction surface)of the HLA molecule and would not directly contact the TCR. Thus,mutations of K71 in DRB1*04:01 are unlikely to induce directalloreactivity. In certain embodiments, suitable engineering sites areevaluated based on their inability to elicit T cell response, such as byin silico modeling, analysis of peptide binding, and/or in vitrocharacterization of T cell responses elicited by engineered HSCs.

Described herein are methods for generating edited HLA alleles that aresufficient to alter antigen binding but do not elicit rejection.Specifically, the edited HLA allele DRB1*04:01^(K71E) is a variant thatis not found in nature, its peptide repertoire and potential foralloreactivity was unknown.

Based on the presently disclosed methodology for identifying HLA targetamino acid positions that may be changed while avoiding rejection by asubject's immune system, Applicants herein show that such amino acidchanges do treat autoimmunity while avoiding rejection. Specifically,Applicants generated transgenic mice expressing either DRB1*04:01 orDRB1*04:01^(K71E) and performed skin transplants between these strains.Skin grafts taken from one DRB1*04:01 mouse and applied to anotherDRB1*04:01 mouse will be accepted by the DRB1*04:01 immune cells asself. However, if the immune cells of DRB1*04:01 mice seeDRB1*04:01^(K71E) as foreign tissue, it will be rejected (and viceversa).

Herein, Applicants experimental results demonstrate that the presentlydisclosed methods of creating engineered HLA alleles based on thesubject's own susceptible HLA allele and HSCs are not rejected. Thedisclosed engineered HLA-DRB1*04:01^(K71E) alleles include onenon-native amino acid substitution in the antigen binding cleft, whereinthe substitution alters binding to at least one antigen (relative thenative susceptible allele) but does not directly affect T-cell receptorinteraction, such as the DRB1*04:01^(K71E) edit, does not inducealloreactivity in native DRB1*04:01 recipients.

More than 100 genetic loci have been associated with RA. However, thestrongest genetic association with RA pathogenesis is with the DRB1 genewithin the major histocompatibility complex, contributing toapproximately 50% of the genetic risk. More specifically, three aminoacid positions (11, 71 and 74; note the aa positions within the HLA arerelative to the mature protein, as presented at the Immuno PolymorphismDatabase-ImMunoGeneTics project/Human Leukocyte Antigen or IPD-IMGT/HLAwebsite, available at website ebi.ac.uk/ipd/imgt/hla) in HLA-DRB1account for most of the association of the HLA DRB1 locus withseropositive RA.

By cloning all the relevant RA-susceptible and RA-resistant alleles andthen performing site-directed mutagenesis on individual amino acids,Applicants demonstrated that mutating position 71 from a K to Econverted the peptide-binding profile to one that was similar to that ofthe resistant HLA allele DRB1*04:02 (below).

Using a peptide competition assay, Applicants identified HLA alleleDRB1*04:01 as possessing the greatest preference for a set ofRA-associated antigens—specifically, post-translationally modified“altered-self” peptides. These altered-self peptides may signal theinitial breach of tolerance in pre-clinical RA. The collection ofaltered self-peptides includes a set of citrullinated peptideneoantigens that are upregulated during infection and inflammation.Human type II collagen is arthritogenic in animal models and in miceCD4+ T cells that initiate arthritis recognize an immunodominant peptidelocated between amino acids 258-272 of collagen. CD4+ T cells thatrecognize the collagen²⁵⁸⁻²⁷² peptide are found in RA joints, and theirpresence in the peripheral blood at disease onset is associated withrapid progression of joint disease and poor responsiveness toconventional synthetic and biologic disease modifying anti-rheumaticdrugs (DMARDs). Applicants discovered that ionic attraction between theacidic K residue in position 71 and a basic E residue in thecollagen²⁵⁸⁻²⁷² peptide enhance peptide binding to DRB1*04:01.

Refractory RA is a more severe form of the disease, in which thesynovium of various joints is maintained in a state of constantinflammation. This state of inflammation is characterized by infiltratesof T cells macrophages, neutrophils, and B cells. The presentlydisclosed compositions, methods, and therapies result in engineered HSCengraftment in the bone marrow that will replenish myeloid APCsexpressing the DRB1*04:01^(K71E) allele.

Without wishing to be limited by theory, unlike native DRB1*04:01, theengineered DRB1*04:01^(K71E) does not bind tightly to collagen²⁵⁸⁻²⁷²and may repel it, resulting in attenuation of the CD4+ T cell responseto this autoantigen. While a population of collagen-specific memory CD4+T cells may persist in the patient, those cells may no longer receivethe necessary TCR signals required to maintain chronic jointinflammation.

The disclosed engineered HSCs will engraft in the bone marrow withindays and begin to generate DRB1*04:01^(K71E)-expressing myeloid cellswithin 10 days. In embodiments where the patients do not undergoimmunosuppressive conditioning prior to administration of the disclosedengineered HSCs, they will retain acquired T and B cell immunity presentbefore the procedure. In embodiments where patients are treated withnon-myeloablative conditioning, using low-dose busulfan, patients mayexperience a brief period of neutropenia (7-10 days; low concentrationof neutrophils, needed for mounting immune responses to infections,especially bacterial) and low platelet counts (20-30 days; possiblyaffecting blood clotting). These phenomena should not belife-threatening and severe adverse events (SAEs) are not expected.

Autoimmune Diseases Treatable with the Presently Disclosed Therapy

Rheumatoid arthritis (RA) is an autoimmune disease characterized byinflammation of the joint capsule synovia, resulting in an infiltrationof macrophages, neutrophils, T cells and B cells that culminates inextensive joint destruction, disability and reduced quality of life. Thepersistent inflammation associated with RA also increases the risk ofdeveloping ischemic heart and respiratory disease, resulting in earlymortality. RA occurs in approximately 1% of the world population, withan estimated 1.3 million affected in the US alone. RA occurs morefrequently in women over the age of 40 and in long-term smokers.Billions of dollars of direct healthcare costs are associated with thetreatment of RA annually, and total annual societal costs of RA (direct,indirect and intangible) are estimated to reach tens of billions ofdollars in the US alone.

Behget's syndrome is a chronic multisystemic inflammatory disordercharacterized by relapsing and recurrent oral ulcers, genital ulcers,skin lesions, uveitis, and broader systemic manifestations, such asarthritis, and gastrointestinal or central nervous system involvement.The disease is categorized as a variable vessel vasculitis with multiplelesions in all sizes of arterial and venous vessels.

Birdshot uveitis (also known as Birdshot chorioretinopathy or Birdshotretinochoroidopathy) is a well-characterized form of autoimmune uveitis(inflammation of the uveal layer of the eye) mostly known for its ovoidlight lesions, which appear ‘shotgun pattern’-like distributed along thevascular arcades in the back of the eye (i.e., the ‘fundus’ of the eyewhere these lesions are visible by photography). Inflammation andextensive depigmentation of the choroid, macular edema, peripheralischemia, degeneration of the retina, and the progressive formation ofthin layer of scar tissue on the retina (“epiretinal membrane”),progressively impair vision in a substantial proportion of patients.Birdshot uveitis typically affects patients over 50 years of age ofWestern-European ancestry, with more women than men affected.

Celiac disease is a chronic immune-mediated enteropathy triggered byexposure to dietary gluten in genetically predisposed individuals (1).In celiac patients, the ingestion of gluten leads to the activation ofboth the innate and adaptive response of the immune system, with asubsequent chronic inflammation that determines changes in the mucosalstructure including villous atrophy, crypt hyperplasia and lymphocyteinfiltration. These changes in structure cause subsequent loss offunction by the intestinal mucosa and the onset of symptoms brought bynutrient malabsorption.

Psoriasis is a chronic inflammatory disease mediated by T lymphocytes,with participation of dendritic cells. Genetic and environmental factorscontribute or are required for the development of overt disease. Thelesions are characterized by erythema and desquamation, configuringdifferent clinical forms, from sharply delimited plaques to diffuseerythroderma. Up to 30% of patients may also have joint involvement,which may result, if untreated, in erosive disease and incapacity. It isconsidered a prevalent disease, affecting 2% of the population inWestern countries.

Narcolepsy was first described by Westphal in 1877 and named by Gélineáuin 1880. After rapid eye movement (REM) sleep was discovered in 1953,several investigators studied sleep onset in patients with narcolepsy.Although healthy individuals typically enter their first REM sleepapproximately 90 min after falling asleep, patients with narcolepsyfrequently go directly into REM sleep at sleep onset. A malfunction ofthe mechanisms that regulate REM sleep can explain some of the symptomsof narcolepsy. Narcolepsy has no known cure at present. Although itssymptoms can be managed with appropriate treatment, lifelong treatmentis required for most patients.

Kawasaki disease (KD) is an acute systemic vasculitis that is a leadingcause of acquired heart disease in children. The pathogenesis of KDremains unknown. It is likely that KD is caused by abnormal immuneresponses to unknown trigger(s) in genetically susceptible children.1The HLA (human leukocyte antigen) genes, known as the most polymorphicgene in vertebrate animals, encode the protein on the cell-surfaceantigen-presenting proteins that is involved in the regulation of theimmune system. The roles of HLA genes have been investigated in severalimmune-mediated vascular diseases, including Behçet disease, KD, andWegener granulomatosis. A recent genome-wide association studydemonstrated the significant association of HLA class II region(HLA-DQB2-DOB) with KD in a Japanese population.

Myasthenia gravis (MG), a rare disorder of the neuromusculartransmission, is increasingly acknowledged as a syndrome rather than asingle disease. In the recent past, there has been an active search fornew antigens in myasthenia gravis, whereas clinical and experimentalstudies have provided new insights into crucial pathways in immuneregulation, which might become the targets of future therapeutics.

Systemic lupus erythematosus (SLE) is a severe autoimmune disease thatinvolves multiple organ systems. Lupus nephritis (LN) is a complicationof SLE and is associated with poor survival and high morbidity. Manygenomic studies have been performed worldwide, and severalhistocompatibility leukocyte antigen (HLA) loci are linked to lupussusceptibility.

Crohn's disease (CD) has been known since 1932, when Crohn et al.reported fourteen cases of terminal ileitis. Crohn's disease is arelapsing inflammatory disease that mainly affects the gastrointestinaltract from mouth to anus. It involves any part of the gastrointestinaltract most commonly the terminal ileum or the perianal region in anon-continuous fashion.

Autoimmune neurology is an expanding field that has seen a hugedevelopment in recent years. Most of this progress is due to thediscovery and characterization of autoantibodies (Ab) directed againstantigens of the peripheral and/or central nervous system, and which areused as biomarkers of these diseases. Some of these Ab have allowed tobetter define already known entities, such as Ab against aquoporin-4(anti-AQP4 Ab) in neuromyelitis optica (NMO).

Type 1 diabetes (T1 D) is a multifactorial autoimmune disease thatresults in destruction of the insulin secreting β cells in the pancreas.Genome wide association studies have identified more than 50 loci linkedto the risk of developing T1 D. However, the inheritance of specifichuman leukocyte antigen (HLA) genes, such as DQ2 and DQ8, is moststrongly linked to disease susceptibility.

Ankylosing spondylitis (AS) is a chronic inflammatory disease thatresults in immune-mediated arthritis of the spine and peripheral joints.The disease is more common in men and symptoms typically begin early inlife. HLA-B*27:05 is strongly associated with AS but B*27:06 and B*27:09are associated with resistance.

Multiple sclerosis (MS) is an autoimmune disease of the brain andcentral nervous system. In MS, the immune system attacks the myelinsheath that covers nerve fibers which can cause permanent damage ordeterioration of the nerves. Susceptibility to MS is associated with theDRB1*15:01 allele.

Shown below at Table 1 are various HLA alleles associated withsusceptibility to the above-described autoimmune diseases. Also shown atTable 1 are target amino acid positions that, when mutated to correspondto the amino acid at the same position in a resistant HLA allele, mayaid in reducing or eliminating at least one symptom associated with theautoimmune disease. Table 1 also discloses specific mutations at thetarget amino acid position for treating the autoimmune disease.

Engineered HLA Molecules

Disclosed herein are various engineered HLA molecules. In someembodiments, the HLA molecule may be selected from one or more of HLA-A,HLA-B, HLA-C, HLA-DPA1, HLA-DPB1, HLA-DQA1, HLA-DQB1, HLA-DRB1,HLA-DRB3, HLA-DRB4, and HLA-DRB5.

In many embodiments the disclosed engineered HLA-A may include mutationsat one or more of polymorphic positions selected from 9, 12, 17, 31, 35,43, 44, 56, 62, 63, 65, 66, 67, 70, 73, 74, 76, 77, 79, 80, 81, 82, 83,90, 95, 97, 99, 102, 105, 107, 109, 114, 116, 127, 142, 144, 145, 149,150, 151, 152, 156, 158, 161, 163, 166, 167, 171, 184, and 186;specifically: 9, 31, 56, 62, 63, 66, 73, 77, 80, 81, 95, 97, 99, 114,116, 150, 152, 156, and 171.

In many embodiments the disclosed engineered HLA-B may include mutationsat one or more of polymorphic positions selected from 4, 9, 11, 12, 24,30, 32, 33, 41, 45, 46, 52, 59, 62, 63, 65, 66, 67, 69, 70, 71, 73, 74,76, 77, 80, 81, 82, 83, 90, 94, 95, 97, 99, 103, 109, 113, 114, 116,131, 143, 145, 147, 152, 156, 158, 162, 163, 166, 167, 171, 177, 178,and 180; specifically: 9, 24, 33, 45, 46, 52, 59, 62, 66, 70, 73, 77,81, 95, 97, 99, 114, 116, 143, 147, 152, 156, 163, 167, 171, and 178.

In many embodiments the disclosed engineered in HLA-C may includemutations at one or more of polymorphic positions selected from 4, 9,11, 12, 24, 30, 32, 33, 41, 45, 46, 52, 59, 62, 63, 65, 66, 67, 69, 70,71, 73, 74, 76, 77, 80, 81, 82, 83, 90, 94, 95, 97, 99, 103, 109, 113,114, 116, 131, 143, 145, 147, 152, 156, 158, 162, 163, 166, 167, 171,177, 178, and 180; specifically: 4, 24, 30, 33, 45, 52, 59, 62, 63, 66,67, 70, 73, 74, 77, 80, 81, 95, 97, 99, 114, 116, 143, 147, 152, 167,and 171.

In many embodiments the disclosed engineered HLA-DQA1 may includemutations at one or more of polymorphic positions selected from 20, 26,34, 40, 41, 44, 46, 47, 48, 50, 52, 53, 54, 55, 61, 64, 66, 69, 75, 76,and 80; specifically: 34, 44, 61, 64, 69, 76, and 80.

In many embodiments the disclosed engineered HLA-DQB1 may includemutations at one or more of polymorphic positions selected from 9, 13,14, 26, 28, 30, 37, 38, 45, 46, 47, 52, 53, 55, 56, 57, 66, 67, 70, 71,74, 75, 77, 84, 85, 86, 87, 89, and 90; specifically: 9, 26, 28, 30, 37,38, 47, 53, 57, 67, 70, 71, 74, 86, 87, and 90.

In many embodiments the disclosed engineered HLA-DPA1 may includemutations at one or more of polymorphic positions selected from 11, 18,28, 30, 31, 50, 72, 73, 83, and 96; specifically: 11, 28, 31, 72, 73,and 96.

In many embodiments the disclosed engineered HLA-DPB1 may includemutations at one or more of polymorphic positions selected from 8, 9,11, 33, 35, 36, 55, 56, 57, 65, 69, 72, 76, 84, 85, 86, 87, and 91;specifically: 9, 11, 33, 35, 36, 55, 56, 65, 69, 72, 76, 84, 87, and 91.

In many embodiments the disclosed engineered HLA-DRB1, HLA-DRB3,HLA-DRB4, and HLA-DRB5 may include mutations at one or more ofpolymorphic positions selected from 9, 10, 11, 12, 13, 14, 16, 25, 26,28, 30, 31, 32, 33, 37, 38, 40, 47, 57, 58, 60, 67, 70, 71, 73, 74, 77,78, 85, and 86; specifically 9, 11, 13, 26, 28, 30, 32, 33, 37, 38, 40,47, 57, 58, 67, 71, 74, 78, 85, and 86.

In many embodiments, the disclosed methods may create one or moreengineered HLA alleles from one or more susceptible HLA alleles selectedfrom:

-   A*02, A*03, A*29;-   B*07, B*08, B*08:01, B*27, B*27:03 B*27:05, B*27:09, B*51, B*54:01,    B*57;-   C*06, C*18;-   DPA1*02:01;-   DPB1*13:01;-   DQ;-   DQA1*02:01, DQA1*03:01, DQA1*05, DQA1*05:01;-   DQB1*02, DQB1*02:01, DQB1*03, DQB1*03:01, DQB1*03:02, DQB1*06:02;-   DR;-   DRB1*01:03, DRB1*04, DRB1*07, DRB1*07:01; DRB1*15, DRB1*15:01;    DRB1*16; DRB1*08, DRB1*08:01, DRB1*11:04.

Disclosed herein are various mutations at target amino acid positionswithin a mature HLA protein sequence. In many embodiments, the mutationsare selected based on the criteria disclosed above. In some embodiments,specific allelic mutations may be selected based on the autoimmunedisease or disorder to be treated. For example, treatments for: Type 1diabetes may include mutations in DQB1*02:01 at A57D (where the nativeA, alanine, at position 57 is mutated to D, aspartic acid) and/orDQB1*03:02 of A57D; rheumatoid arthritis may include one or moremutations in DRB1*04:01 of L67l, Q70D, L67l+Q70D, K71E, K71R, L67F,A74L, L67F-A74F, G86V, G86M, G86L, G86F, and A74E; in DRB1*04:05 ofR71E, in DRB1*01:01 of L67l, Q70D, R71E, V85A, and G86V, in DRB1*04:03of R71E, in DRB1*04:04 of R71E, in DRB1*04:08 of R71E; multiplesclerosis may include one or more mutations in DRB1*15:01 of F47Y, A71R,A71R-V86G, V86L, and V86F; celiac disease may include one or moremutation in DQB1*02:01 of K71E, and K71T; neuromyelitis optica mayinclude one or more mutation in DRB1*03:01 of V86L and V86M; Behçet'ssyndrome may include one or more mutation in B*51; psoriasis my includeone or more mutations in C*06; B*57, and C*06; C*18, A*02.

Methods of Treatment

The present disclosure includes methods of treating or preventing anautoimmune disease by administering engineered APCs and/or APCprecursors, i.e., engineered HSCs. In contrast to, for example, T celltherapies, the engineered cell compositions disclosed herein areprovided to reduce or prevent T cell-mediated rejection responses,rather than elicit them. As such, the engineered compositions provide arelatively broad therapeutic window, while targeting the subject'sspecific condition. In certain embodiments, the methods compriseadministration of a therapeutically effective amount of the engineeredHSCs.

In certain embodiments, subjects receive 1×10⁶, 2×10⁶, 3×10⁶, 4×10⁶,5×10⁶, for example 1-5 million, or more engineered, autologous, HSCs perkg of body weight, such as by intravenous administration, in one or moredoses, over one or more days.

The methods of the present disclosure include the production andadministration of engineered HSCs as described. In certain embodiments,including presently certain embodiments, the engineered HSCs areautologous to the subject to be treated. Accordingly, some embodimentsinclude isolating HSCs or HSC precursors from the subject, ex vivoengineering of the isolated HSCs, optional selection and/or expansion ofthe engineered HSCs, and administration of the engineered autologousHSCs to the subject.

HSCs or precursors can be isolated from subjects by methods known in theart. For example, PBMCs and/or bone marrow cells can be mobilized,isolated, and HSCs purified based on expression of CD34. HSCsubpopulations can be selected based on expression of additionalantigens as desired. Additionally, or alternatively, HSCs can beproduced from precursor cells, such as pre-harvested stem cells orde-differentiated cells of the subject, as known in the art. Althoughautologous HSCs are presently certain, the present disclosure is notlimited to autologous HSCs. For example, in certain embodiments,non-autologous (donor) HSCs engineered to express a desired HLA withoutexpressing proteins capable of eliciting non-self-responses areprovided.

Certain embodiments of the methods provided herein also includepre-conditioning, such as non-myeloablative conditioning, and/orpost-treatment interventions, such as to promote engraftment of theengineered HSCs. Additionally, or alternatively, HSC engineeringaccording to the present disclosure can be performed in vivo, such as byadministration of viral vectors encoding, inter alia, expressedautoimmunity resistance alleles and/or gene editing constructs asdescribed herein. Moreover, although reference is primarily made hereinto single engineered HSC populations, multiple HSC compositions havingdiscrete HLA allelic modifications may also be provided, separately orsequentially, such as in instances of multi-allelic autoimmune disease.

HLA Allele Engineering

The present disclosure includes systems, constructs, and techniques forgene editing, and the application of same to provide resistance toautoimmunity. In particular, certain embodiments of the presentdisclosure include constructs, systems, and vectors for HLA alleleengineering as disclosed herein.

Many gene editing systems are available, suitable, andwell-characterized in the art. For example, in certain embodiments,CRISPR-Cas systems containing a DNA-targeting polynucleotidecomplementing the HLA allele to be modified and a CRISPR-associatednuclease, such as Cas9, are provided. Related CRISPR-Cas9 systems fortreatment of RNA are disclosed in PCT/US2018/029302, published asWO2019200635, hereby incorporated by reference herein in its entirety.

In other embodiments, CRISPR systems containing, for example, CasX,Cas12a, Cas13, or MAD7 are provided for HSC HLA allele engineering, forexample as in PCT/US2019/043066, published as WO/2020/023529,PCT/US2018/028919, published as WO/2018/195545, etc. Certain CRISPRsystems can be selected on the basis of protospacer-adjacent motif (PAM)specificity, allowing targeting of almost all genomic sequences,on-target selectivity, efficiency in human HSCs, and otherconsiderations. In alternative embodiments, TAL effector nucleases(TALENs) or zinc finger nucleases (ZFNs) are employed for HLA alleleengineering as disclosed at Nucleic Acid Res. 2011 Sep. 1; 39(17):7879).

In further embodiments, HLA allele engineering is performed with fusionproteins, such as enzymatically inactive dCas9-based fusion proteins.These systems combine the programmable DNA-targeting capabilityassociated with CRISPR with additional on-target selectivity and/orfunctional capabilities of other gene engineering platforms. Forexample, in certain embodiments, HLA allele engineering is performedwith systems including a Cas-CLOVER fusion, as described inPCT/US2015/036226.

In additional embodiments, including certain embodiments, HLA alleleengineering is performed with a nucleobase editing system. For example,certain embodiments provide an HLA-allele targeting polynucleotide and afusion protein comprising dCas9 and a nucleobase editing enzyme, such asa deaminase. Such embodiments advantageously result in generation ofspecific point mutations sufficient to alter the amino acid encoded atthe targeted HLA allele codon without resulting in or requiring DNAdouble-strand breakage and repair.

The principles of design for CRISPR-Cas systems and vectors for same arewell known in the art, and in the present context essentially requireonly selection of a sequence complementary to the portion of the HLAallele to be engineered. The same is true with respect to CRISPR-Casfusion-based systems including the examples described. The production ofgenetic engineering platforms involving protein-based DNA targeting,such as TALENs and zinc fingers, is also well-characterized, andsuitable such systems for use with the present disclosure can begenerated with no more than routine procedures and experimentation.

In additional and alternative embodiments, the HLA allele engineeringsystems include a homologous repair template. For example, in certainembodiments, the entire gene for the susceptible HLA allele, within theMHC locus, can be excised and replaced with the engineered HLA allele.In many embodiments, the gene coding for the susceptible HLA allele maybe disrupted by insertion of the engineered HLA allele, which may be asan uninterrupted nucleic acid with the engineered HLA allele's cDNAsequence.

HLA allele engineering, and the systems therefore, according to thepresent disclosure can also include, for example, vectors, such asretroviral vectors for expression of the HLA allele engineeringconstructs disclosed. Transient transfection techniques and systemstherefore can also be applied. Accordingly, the present disclosure isnot limited by or to specific HLA allele engineering constructs orsystems. In certain embodiments, including some certain embodiments, HLAallele engineering according to the table below is provided.

TABLE 1 Target Amino Autoimmune Disease Engineered Allele Acid MutationType 1 Diabetes DQB1*02:01 57 A57D DQB1*03:02 57 A57D RheumatoidArthritis DRB1*04:01 67 L67I, 70 Q70D 71 K71E; K71R, 67 L67F 74 A74L 74A74E 86 G86L; G86F; G86V; G86M DRB1*04:05 71 R71E DRB1*01:01 67 L67I 70Q70D 71 R71E 85 V85A 86 G86A DRB1*04:03 71 R71E DRB1*04:04 71 R71EDRB1*04:08 71 R71E Ankylosing Spondylitis B*27:05 116 D116H MultipleSclerosis DRB1*15:01 47 F47Y 71 A71R 86 V86L; V86F Celiac DiseaseDQB1*02:01 71 K71E; K71T Neuromyelitis Optica DRB1*03:01 86 V86L; V86MBehçet’s Syndrome B*51 Psoriasis C*06 B*57 C*18 A*02

Engineered Hematopoietic Cells

Autologous immune cells may be engineered using various systems asdisclosed herein, for example cells may be engineered to carry andexpress engineered HLA genes and molecules with various viral vectorsand/or nucleases capable of genomic editing. Various protocols wellknown to those of skill in the art may allow for screening of thegenomes of manipulated cells to assess the frequency and/or position ofviral insertions, double strand breaks in DNA (DSBs) or otherpotentially mutagenic events (Li H, Haurigot V, Doyon Y, et al. In vivogenome editing restores haemostasis in a mouse model of haemophilia.Nature. 475(7355):217-21, 2011). In many embodiments, the systems may beuseful in removing and or preventing expression of the susceptible HLAallele, as well as inserting the engineered HLA allele into the samelocus. In many embodiments, the engineered HLA allele is expressed froma cDNA sequence. Particular specific cDNA sequences of susceptible HLAalleles and engineered HLA alleles are provided at FIG. 30 , and SEQ IDNOs:59-96.

Therapeutically relevant levels of genetically modified engineeredhematopoietic stem cells needed to effect clinical outcomes may be morereadily achieved by expansion of large populations of cells ex vivo andreintroduction(s) into the patient.

Definitions

The following terms and phrases include the meanings provided below. Theprovided definitions are intended to aid in describing particularembodiments, and are not intended to limit the claimed compositions,methods, compounds, systems, and therapies. Unless otherwise defined,all technical and scientific terms used herein have the same meaning ascommonly understood by one of ordinary skill in the art to which thisdisclosure belongs. If there is an apparent discrepancy between theusage of a term in the art and its definition provided herein, thedefinition provided within the specification shall prevail.

The term “about” or “approximately” means an acceptable error for aparticular value as determined by one of ordinary skill in the art,which depends in part on how the value is measured or determined. Incertain embodiments, the term “about” or “approximately” means within 1,2, 3, or 4 standard deviations. In certain embodiments, the term “about”or “approximately” means within 30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%,5%, 4%, 3%, 2%, 1%, 0.5%, or 0.05% of a given value or range. Wheneverthe term “about” or “approximately” precedes the first numerical valuein a series of two or more numerical values, it is understood that theterm “about” or “approximately” applies to each one of the numericalvalues in that series.

“Amino acid identity,” “residue identity,” “identity,” and the like, asused herein refers to the structure of the functional group (R group) onthe poly peptide backbone at a given position. Naturally occurring aminoacid identities are (name/3-letter code/one-letter code): alanine/ala/A;arginine/arg/R; asparagine/asn/N; aspartic acid/asp/D; cysteine/cys/C;glutamine/gln/Q; glutamic acid/glu/E; glycine/gly/G; histidine/his/H;isoleucine/ile/I; leucine/leu/L; lysine/lys/K; methionine/met/M;phenylalanine/phe/F; proline/pro/P; serine/ser/S; threonine/thr/T;tryptophan/trp/W; tyrosine/tyr/Y; and valine/val/V. Amino acidpositions, as used herein to designate a position on an HLA moleculereference the mature protein sequence as provided at websiteebi.ac.uk/ipd/imgt/hla. Thus, for example, DRB1*04:01^(K71E), refers toposition 71 in the mature protein of allele *04:01 of DRB1, wherein thenative identity is lysine, K, and the non-native, edited identity isglutamic acid, E.

“Autoimmune disease, disorder, or condition” refers to a disease,disorder, or condition in which the immune system produces an immuneresponse (e.g., a B cell or a T-cell response) against an endogenousantigen, leading to injury one or more tissues. Such diseases include,but are not limited to, rheumatoid arthritis (RA), celiac disease,diabetes mellitus type 1, systemic lupus erythematosus (SLE), multiplesclerosis (MS), myelin oligodendrocyte glycoprotein antibody disorders(MOGAD), myasthenic syndromes and neuromyelitis optica (NMO), ankylosingspondylitis, Behget's syndrome, Birdshot uveitis, narcolepsy, narcolepsytype 1 (NT1; previously termed narcolepsy with cataplexy), Kawasakidisease, Crohn's disease, psoriasis, dermatomyositis (DM), Addison'sdisease, irritable-bowl syndrome (IBS), Graves' disease,Henoch-Schönlein purpura (HSP), sarcoidosis, Sjögren's syndrome,eosinophilic granulomatosis with polyangiitis, Hashimoto's disease,idiopathic thrombocytopenic purpura, polymyositis (PM), paraneoplasticneurological syndromes (PNS), autoimmune encephalitis, lupus nephritis(LN), myasthenia gravis (MG), psoriatic arthritis, graft rejection,graft-versus-host disease (GVHD), an unwanted delayed-typehypersensitivity reaction, T-cell mediated pulmonary disease, neuritis,vitiligo, autoimmune pancreatitis, inflammatory bowel diseases,ulcerative colitis, glomerulonephritis, scleroderma, autoimmune thyroiddiseases, asthma, autoimmune uveoretinitis, pemphigus vulgaris,pulmonary fibrosis or idiopathic pulmonary fibrosis, primary biliarycirrhosis, and pernicious anemia. When used herein, the terms “disease,”“disorder,” and “condition” are interchangeable.

“HLA” or “human leukocyte antigen” refers to human gene that encodes amajor histocompatibility complex (MHC) protein on the surface of cellsthat are responsible for regulation of the immune system. “HLA-I” or“HLA class I” refers to human MHC class I gene including HLA-A, HLA-B,HLA-C, HLA-E, HLA-F, HLA-G, and β2-microglobulin loci. “HLA-II” or “HLAclass II” refers to human MHC class II gene including HLA-DPA1,HLA-DPB1, HLA-DQA1, HLA-DQB1, HLA-DRA1, HLA-DRB1, HLA-DRB3, HLA-DRB4,HLA-DRB5, HLA-DM, HLA-DOA, and HLA-DOB loci.

“Intravenous” administration refers to administering a drug or therapy,for example one or more of the disclosed engineered HSCs into a vein ofa patient, e.g. by infusion (slow therapeutic introduction into thevein) for therapeutic purposes. “Infusion” or “infusing” refers to theintroduction of a drug, therapy, and/or solution into the body of apatient through a vein for therapeutic purposes. Generally, this may beaccomplished via an intravenous (IV) bag.

An “intravenous bag” or “IV bag” is a bag that can hold a solution whichcan be administered via the vein of a patient. In one embodiment, thesolution may be a saline solution (e.g. about 0.9% or about 0.45% NaCl),or any therapeutically useful solution for administration of thedisclosed engineered HSCs.

By “co-administering” is meant intravenously administering two (or more)drugs during the same administration, rather than sequential (i.e. oneafter the other) infusions of the two or more drugs. Generally,co-administration may involve combining the two (or more) drugs into thesame IV bag, or adding the second drug to an I.V. bag comprising thefirst drug, prior to co-administration thereof.

The term “amelioration” as used herein refers to any improvement of adisease state (for example improvement of a symptom of an autoimmunedisease, for example a symptom of rheumatoid arthritis) of a patientsuffering therefrom, by the administration of one or more treatments,drugs, and/or compositions, according to the present disclosure, to suchpatient or subject in need thereof. Such an improvement may be seen as aslowing down of the progression, or a cessation of the progression, ofthe disease of the patient, a decrease in the frequency, duration,and/or severity of any symptom, and/or an increase in frequency orduration of disease symptom-free periods or a prevention of impairmentor disability due to the disease.

“Antigen” refers to a compound, composition, substance, protein,peptide, nucleic acid, nucleo-peptide, etc., whether native, modified,or synthetic, that can stimulate the production of antibodies or aT-cell response in an animal, including compositions that are injectedor absorbed into an animal or modified by an animal. As used herein, anantigen may be defined by its ability to bind within the antigen bindingcleft of a native or engineered HLA molecule. In some aspects, anantigen may react with one or more products of specific humoral orcellular immune system. The term “antigen” includes all relatedantigenic epitopes and antigenic determinants.

“Antigen Presenting Cell,” (APC), refers to a cell that can process andpresent antigenic compounds, including peptides, in association withclass I or class II MHC molecules to a T-cell. In many cases, the APCcan deliver a co-stimulatory signal necessary for T-cell activation.Typical APCs include monocytes, macrophages, dendritic cells, B cells,thymic epithelial cells, and vascular endothelial cells.

“Antigen binding region,” “antigen binding cleft,” “antigen bindinggroove,” “antigen cleft,” and the like refer to the region of the HLAmolecule that interacts with and binds the antigen presented by the HLA.As discussed in Nguyen, A. et al., “The pockets guide to HLA class Imolecules,” Biochemical Society Transactions (2021) 49 2319-2331, whichis incorporated herein, the HLA-I peptide binding cleft is closed at theN and C termini (restricting the length of peptide antigens to about8-10 amino acids), while the ends of the HLA-II cleft is open, allowingfor longer peptide antigens (for example>13 amino acids in length). K.J. Smith et al., “Crystal Structure of HLA-DR2 (DRA*0101, DRB1*1501)Complexed with a Peptide from Human Myelin Basic Protein,” Vol. 188, No.8, Oct. 19, 1998, 1511-1520, which is incorporated herein, discussbinding cleft and pocket structures in HLA Class II molecules. Ingeneral, specific binding pockets bind specific components of an antigenbound within the antigen binding cleft. As used herein, the ‘bottom’ ofthe cleft may be the surface of the cleft nearest the core of themolecule and farthest from the TCR interface, the cleft may have sidesextending generally upward from the bottom toward the TCR interface. Inmost embodiments, the antigen is a peptide and the components are aminoacids. Three-dimensional structures of HLA molecules are available tothe skilled artisan for reference (for example atebi.ac.uk/ipd/imgt/hla), allowing for identification of the antigenbinding region for any HLA molecule.

cDNA (complementary DNA) are poly nucleic acids lacking internal,non-coding segments (introns) and regulatory sequences that determinetranscription. cDNA is synthesized in the laboratory by reversetranscription from messenger RNA extracted from cells.

The terms “dosage” or “dose” as used herein denote any form of theactive ingredient formulation that contains an amount sufficient toproduce a therapeutic effect with a single administration.

The phrase “therapeutically effective amount” means an amount of a drug,composition, compound, treatment, or therapy of the present disclosurethat alone, or in combination with other therapies, (i) treats theparticular disease, condition, or disorder, (ii) attenuates,ameliorates, or eliminates one or more symptoms of the particulardisease, condition, or disorder, or (iii) prevents or delays, the onsetof one or more symptoms of the particular disease, condition, ordisorder described herein. The term can encompass an amount thatimproves overall therapy, reduces, or avoids symptoms or causes ofdisease, or enhances the therapeutic efficacy or synergizes with anothertherapeutic agent. In the case of the targeted autoimmunity, thetherapeutically effective amount of the drug, composition, compound,treatment, or therapy may reduce the number of reactive or active immunecells, such as T-cells; reduce inflammation; inhibit (i.e., slow to someextent and preferably stop) immune-based attack or degradation of cells,tissues, or organ; and/or partially or fully relieve one or more of thesymptoms associated with the autoimmune response.

“Immune response” refers to a response of a cell of the immune system,such as a B cell, or a T-cell, to a stimulus. In one embodiment, theresponse is specific for a particular antigen (an “antigen-specificresponse”). In another embodiment, an immune response is a T-cellresponse.

“Autoimmune response” refers to an immune response directed against anauto- or self-antigen. In many cases, the autoimmune response is aresult of self-reactive T cells, which recognize one or more auto- orself-antigens. The immune system ordinarily functions to directprotective immune responses against microorganisms and other harmfulforeign materials. In an autoimmune response, antigens present in apatient's own tissues become targets for autoreactive immune responsesthat cause cell, tissue, or organ deterioration, destruction, ordysfunction.

The term “mammal” includes, but is not limited to, humans, mice, rats,guinea pigs, monkeys, dogs, cats, horses, cows, pigs and sheep.

A “patient” or “subject” includes a mammal or animal, such as a human,cow, horse, sheep, lamb, pig, chicken, turkey, quail, cat, dog, mouse,rat, rabbit, or guinea pig. The animal can be a mammal such as anon-primate or a primate (e.g., monkey and human). In one embodiment, apatient is a human, such as a human infant, child, adolescent, or adultof any or indeterminant sex.

“Pharmaceutically acceptable composition” is an organic or inorganicsolution for maintaining or supporting the viability of a mammaliancell.

“Prevention” as used herein means the avoidance of the occurrence or ofthe re-occurrence of a disease, disorder, or condition as specifiedherein, by the administration of a composition, compound, treatment, ortherapy according to the present disclosure to a subject in needthereof.

“Recombinant” refers to a nucleic acid or polypeptide that has asequence that is not typically found or expressed in a patient or has asequence that is the result of artificial manipulation, such as mutationof one or more nucleic acids or amino acids. Artificial manipulation maybe accomplished by chemical synthesis or, more commonly, by the editing(insertion, deletion, mutation, etc.) of isolated segments of nucleicacids, e.g., by genetic engineering techniques.

Similarity between amino acid or peptide sequences is expressed in termsof the similarity between two sequences, otherwise referred to assequence identity. Sequence identity is frequently measured in terms ofpercentage identity (percentage of identical residues for peptides orbases for nucleic acids; or similarity or homology); the higher thepercentage, the more similar the two sequences are. Complete identity is100% identical over a given sequence, for example 50, 100, 150, or 200bases or residues.

The term “specifically binds” is “antigen specific,” is “specific for,”“selective binding agent,” “specific binding agent,” “antigen target” oris “immunoreactive” with an antigen refers to an molecule or polypeptidethat binds a target antigen with greater affinity than other antigens ofsimilar sequence. It is contemplated herein that the antigenspecifically binds HLA molecules at the surface of an APC.

“Subject in need,” “patient” or those “in need of treatment” includethose already with existing disease (i.e. autoimmune disease, forexample, without limitation, rheumatoid arthritis (RA), celiac disease,diabetes mellitus type 1, systemic lupus erythematosus (SLE), multiplesclerosis (MS), myelin oligodendrocyte glycoprotein antibody disorders(MOGAD), myasthenic syndromes and neuromyelitis optica (NMO), ankylosingspondylitis, Behget's syndrome, Birdshot uveitis, narcolepsy, narcolepsytype 1 (NT1; previously termed narcolepsy with cataplexy), Kawasakidisease, Crohn's disease, psoriasis, dermatomyositis (DM), Addison'sdisease, irritable-bowl syndrome (IBS), Graves' disease,Henoch-Schönlein purpura (HSP), sarcoidosis, Sjögren's syndrome,eosinophilic granulomatosis with polyangiitis, Hashimoto's disease,idiopathic thrombocytopenic purpura, polymyositis (PM), paraneoplasticneurological syndromes (PNS), autoimmune encephalitis, lupus nephritis(LN), myasthenia gravis (MG), psoriatic arthritis, graft rejection,graft-versus-host disease (GVHD), an unwanted delayed-typehypersensitivity reaction, T-cell mediated pulmonary disease, neuritis,vitiligo, autoimmune pancreatitis, inflammatory bowel diseases,ulcerative colitis, glomerulonephritis, scleroderma, autoimmune thyroiddiseases, asthma, autoimmune uveoretinitis, pemphigus vulgaris,pulmonary fibrosis or idiopathic pulmonary fibrosis, primary biliarycirrhosis, and pernicious anemia), as well as those at risk of orsusceptible to the disease. The terms also include human and othermammalian subjects that receive either prophylactic or therapeutictreatments as disclosed herein.

“Tolerance” refers to a diminished or absent capacity to make a specificimmune response to an antigen. Tolerance is often produced as a resultof contact with an antigen in the presence of a two domain MHC molecule,as described herein. In one embodiment, a B cell response is reduced ordoes not occur. In another embodiment, a T-cell response is reduced ordoes not occur. Alternatively, both a T-cell and a B cell response canbe reduced or not occur.

The terms “treat,” “treating,” and “treatment” refer to eliminating,reducing, suppressing, or ameliorating, either temporarily orpermanently, either partially or completely, a clinical symptom,manifestation or progression of an event, disease or conditionassociated with immune disorders and diseases described herein. As isrecognized in the pertinent field, methods and compositions employed astherapies may reduce the severity of a given disease state but need notabolish every manifestation of the disease to be regarded as useful.Similarly, a prophylactically administered treatment need not becompletely effective in preventing the onset of a condition toconstitute a viable prophylactic method or agent. Simply reducing theimpact of a disease (for example, as disclosed herein, reducinginflammation, T-cell activation, etc. and/or reducing the number orseverity of associated symptoms, or by increasing the effectiveness ofanother treatment, or by producing another beneficial effect), orreducing the likelihood that the disease will occur or worsen in asubject, is sufficient. One embodiment of the present disclosure isdirected to a method for determining the efficacy of treatmentcomprising administering to a patient therapeutic treatment in anamount, duration, and repetition sufficient to induce a sustainedimprovement over pre-existing conditions, or a baseline indicator thatreflects the severity of the particular disorder.

As used herein, the terms “protein” and “polypeptide” are usedinterchangeably to designate a series of amino acid residues, connectedto each other by peptide bonds between the alpha-amino and carboxygroups of adjacent residues. The terms “protein”, and “polypeptide”refer to a polymer of amino acids, including modified amino acids (e.g.,phosphorylated, glycated, glycosylated, etc.) and amino acid analogs,regardless of its size or function. “Protein” and “polypeptide” areoften used in reference to relatively large polypeptides, whereas theterm “peptide” is often used in reference to small polypeptides, butusage of these terms in the art overlaps. The terms “protein” and“polypeptide” are used interchangeably herein when referring to a geneproduct and fragments thereof. Thus, exemplary polypeptides or proteinsinclude gene products, naturally occurring proteins, homologs,orthologs, paralogs, fragments and other equivalents, variants,fragments, and analogs of the foregoing.

An amino acid within an HLA molecule may be substituted to create anengineered HLA molecule. The amino acid (aa or a.a.) residue can bereplaced by a residue having similar physiochemical characteristics,that is a ‘conservative substitution’—e.g., substituting one aliphaticresidue for another (such as Ile, Val, Leu, or Ala for one another), orsubstitution of one polar residue for another (such as between Lys andArg; Glu and Asp; or Gln and Asn). Other such conservativesubstitutions, for example based on size, charge, polarity,hydrophobicity, chain rigidity/orientation, etc., are well known in theart of protein engineering. Polypeptides comprising conservative aminoacid substitutions can be tested in any one of the assays describedherein to confirm that a desired activity, e.g. binding, specificity,and/or function of a native or reference polypeptide is achieved.

Amino acids can be grouped according to similarities in the propertiesof their side chains (in A. L. Lehninger, in Biochemistry, second ed.,pp. 73-75, Worth Publishers, New York (1975)): (1) non-polar: Ala (A),Val (V), Leu (L), Ile (I), Pro (P), Phe (F), Trp (W), Met (M); (2)uncharged polar: Gly (G), Ser (S), Thr (T), Cys (C), Tyr (Y), Asn (N),Gln (Q); (3) acidic: Asp (D), Glu (E); (4) basic: Lys (K), Arg (R), His(H). Alternatively, naturally occurring residues can be divided intogroups based on common side-chain properties: (1) hydrophobic: leucine,Met, Ala, Val, Leu, Ile; (2) neutral hydrophilic: Cys, Ser, Thr, Asn,Gln; (3) acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues thatinfluence chain orientation: Gly, Pro; (6) aromatic: Trp, Tyr, Phe.Non-conservative substitutions will entail exchanging a member of one ofthese classes for another class. Particular conservative substitutionsinclude, for example; Ala into Gly or into Ser; Arg into Lys; Asn intoGln or into His; Asp into Glu; Cys into Ser; Gln into Asn; Glu into Asp;Gly into Ala or into Pro; His into Asn or into Gln; Ile into Leu or intoVal; Leu into Ile or into Val; Lys into Arg, into Gln or into Glu; Metinto Leu, into Tyr or into Ile; Phe into Met, into Leu or into Tyr; Serinto Thr; Thr into Ser; Trp into Tyr; Tyr into Trp; and/or Phe into Val,into Ile or into Leu.

“T-cell” refers to immune cells that have matured in the thymus. Anactivated T-cell is a T-cell that has left G₀ and is synthesizing DNA,upregulating CD25, and/or up-regulating CD

“T-cell receptor,” or “TCR,” as used herein refers to a cell surfaceprotein on T-cells that recognizes/interacts with an HLA molecule on anAPC.

“T-cell receptor:HLA binding interface,” T-cell receptor bindinginterface,” “TCR:HLA binding interface,” “TCR:HLA interface,” as usedherein refers to the surface of the TCR and the surface of the HLAmolecule in close proximity during TCR:HLA binding, in most cases, theTCR:HLA binding interface does not include amino acids within theantigen binding cleft of the HLA that are not in direct contact with theTCR.

“Variant,” as used herein refers to a polypeptide, nucleic acid, gene,sequence, or molecule that is substantially homologous to a naturallyoccurring or reference member, but which is different from that of thenative or reference member because of one or a plurality of deletions,insertions, substitutions, molecules, expression levels, etc. Variantpolypeptide-encoding DNA sequences encompass sequences that comprise oneor more additions, deletions, or substitutions of nucleotides whencompared to a native or reference DNA sequence, but that encode avariant protein or fragment thereof. A wide variety of cloning,PCR-based site-specific mutagenesis, and genomic editing approaches areknown in the art, and can be applied by the ordinarily skilled artisan.

Variant HLA genes and molecules include those naturally occurringvariants, as listed at the IPD-IMGT/HLA Database (websiteebi.ac.uk/ipd/imgt/hla; the “IPD Database”). For example, HLA-A Variantsinclude all HLA-A alleles from *01 through to *80, listed at the IPDDatabase.

Variant amino acid or nucleic acid sequences can be at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, or more, identical to a native orreference sequence. The degree of homology (percent identity) between anative and variant sequence can be determined, for example, by comparingthe two sequences using freely available computer programs commonlyemployed for this purpose on the world wide web (e.g., BLASTp or BLASTnwith default settings).

Alterations of the native amino acid sequence can be accomplished by anyof a number of techniques known to one of skill in the art. Mutationscan be introduced, for example, at particular loci by synthesizingoligonucleotides containing a mutant sequence, flanked by restrictionsites enabling ligation to fragments of the native sequence. Followingligation, the resulting reconstructed sequence encodes an analog havingthe desired amino acid insertion, substitution, or deletion.Alternatively, oligonucleotide-directed site-specific mutagenesisprocedures can be employed to provide an altered nucleotide sequencehaving particular codons altered according to the substitution,deletion, or insertion required. Techniques for making such alterationsare very well established and understood by those of skill in the art.

“Nucleic acid” or “nucleic acid sequence” refers to any molecule,preferably a polymeric molecule, incorporating units of ribonucleicacid, deoxyribonucleic acid or an analog thereof. The nucleic acid canbe either single-stranded or double-stranded. A single-stranded nucleicacid can be one nucleic acid strand of a denatured double-stranded DNA.Alternatively, it can be a single-stranded nucleic acid not derived fromany double-stranded DNA. In one aspect, the nucleic acid can be DNA. Inanother aspect, the nucleic acid can be RNA. Suitable DNA can include,e.g., genomic DNA, cDNA, or vector DNA. Suitable RNA can include, e.g.,mRNA.

“Expression” as used herein, refers to cellular processes involved inproducing, displaying (e.g., on or at a cell's surface/outer membrane),or secreting RNA and proteins including where applicable, but notlimited to, for example, transcription, transcript processing,translation and protein folding, modification and processing. Expressioncan refer to the transcription and stable accumulation of sense (e.g.,mRNA) or antisense RNA derived from a nucleic acid fragment or fragmentsand/or to the translation of mRNA into a polypeptide.

“Vector” refers to a nucleic acid molecule which is capable oftransporting another nucleic acid linked, typically covalently usinggene engineering methods, thereto. One type of vector is a “plasmid,”which refers to circular double-stranded DNA into which an additionalDNA segment can be ligated. Another type of vector is a phage vector.Yet another type of vector is a viral vector, where an additional DNAsegment can be ligated into the viral genome. Certain vectors arecapable of autonomous replication in a host cell into which they areintroduced (for example, bacterial vectors having a bacterial origin ofreplication and episomal mammalian vectors). Other vectors (for example,non-episomal mammalian vectors) can be integrated into the genome of ahost cell upon introduction into the host cell, and thus are replicatedalong with the host genome. In addition, certain vectors are capable ofdirecting expression of genes to which they are operatively linked. Suchvectors are referred to herein as “recombinant expression vectors” orsimply “expression vectors.” In general, expression vectors useful inrecombinant DNA techniques are often in the form of plasmids. In thepresent specification, “plasmid” and “vector” may be usedinterchangeably as the plasmid is the most commonly used form amongvectors.

“Engineered” as used herein may refer to the aspect of having beenmanipulated by human intervention. Disclosed herein are engineeredcells, HSCs, peptides, polypeptides, proteins, molecules, HLA proteins,nucleic acids, genes, etc. In one example, an HLA protein is consideredto be “engineered” when at least one aspect of the polypeptide, e.g.,its sequence, has been intentionally manipulated by human intervention(directly or indirectly) to differ from the aspect as it exists in apatient/subject or in nature. As is common practice and is understood bythose in the art, progeny of an engineered cell are typically stillreferred to as “engineered” even though the actual manipulation wasperformed on a prior entity. In contrast, “native” or “wild-type” asused herein refers to un-engineered and/or un-modified cells, genes,proteins, nucleic acids, nucleic acid sequences, alleles, and amino acidsequences, and portions thereof.

Abbreviations: ACR, American College of Rheumatology; ADA, Anti-drugAntibody; AE, Adverse Event; ANC, Absolute Neutrophil Count; APC,Antigen Presenting Cell; AUC, Area Under the Concentration-Time Curve;DMARD, Disease-Modifying Anti-Rheumatic Drug; CBC, Complete Blood Count;cGCP, Current Good Clinical Practices; CD, Cluster of Differentiation;CMP, Complete Metabolic Panel; cGMP, Current Good ManufacturingPractices; cGTP, Current Good Tissue Practices; DC, Dendritic cell; DM,dermatomyositis; DMSO, Dimethyl Sulfoxide; DRB1*04:01, HLA DR Beta 1chain of HLA, 04:01 Allele; E, Glutamic Acid; EBMT, European Bone MarrowTransplant Registry; G-CSF, Granulocyte Colony Stimulating Factor; GVHD,graft-versus-host disease; GWAS, Genome Wide Association Study; HLA,Human Leukocyte Antigen; HLA-DRB1, Human Leukocyte Antigen— DR Beta 1;HSA, Human Serum Albumin; HSC, Hematopoietic Stem Cell; HSP,Henoch-Schönlein purpura; IBS, irritable-bowl syndrome; IL, Interleukin;IV, Intravenous; K, Lysine; LN, lupus nephritis; MG, myasthenia gravis;MHC II, Major Histocompatibility Complex Class II (HLA in Humans);MOGAD, myelin oligodendrocyte glycoprotein antibody disorders; MS,multiple sclerosis; MTX, Methotrexate; NIS, National Inpatient SampleDataset; NMO, neuromyelitis optica; NSAIDs, Non-SteroidalAnti-Inflammatory Drugs; NT1, narcolepsy type 1; PNS, paraneoplasticneurological syndromes; PM, polymyositis; RA, rheumatoid arthritis; SC,Subcutaneous; SAE, Serious Adverse Event; SLE, systemic lupuserythematosus; TCR, T Cell Receptor; TNF, Tumor Necrosis Factor.

“Susceptible HLA allele,” “susceptibility allele,” and the like as usedherein refers to a given HLA allele that is associated withsusceptibility to one or more autoimmune diseases in a given population.

“Resistant HLA allele,” “resistance allele,” and the like as used hereinrefers to a given HLA allele that is associated with resistance to oneor more autoimmune diseases in a given population.

“Genome” as used herein refers to all genetic information of anorganism, including both coding (i.e. genes) and noncodingdeoxyribonucleic acids (DNA). “Genomic sequence” is the nucleotidesequence of the genome's DNA. “native genome” as used herein refers toreferss to the original genomic sequence of an individual, such as asubject or patient, before any modification or engineering as describedherein.

Certain terms referring to like subject matter may be usedinterchangeably herein. For example, reference to HLA proteins encodedby a specific allele of a specific gene may be identified with referenceto the HLA allele. Similarly, specific codons in an HLA allele may beidentified with reference to the amino acid encoded thereby. Forexample, a specific position in the amino acid sequence of an HLAprotein encoded by an HLA allele may be identified by reference to thecorresponding position in the HLA allele, and vice versa.

EXAMPLES Example 1—Materials and Methods Cell Lines

Briefly, cDNA expression constructs were obtained either by cloning theallele of interest directly from cells expressing that allele, or byobtaining ‘gBlock’ sequences (based on IPD-IMGT/HLA Database sequences,available at website ebi.ac.uk/ipd/imgt/hla/) from Integrated DNATechnologies (Coralville, Iowa). Various gBlock sequences, with RE sitesare shown at FIG. 30 . For testing, cDNA was cloned into a murine stemcell virus (MSCV) plasmid for retroviral transduction and expression.Various alleles were individually packaged as retrovirus by transienttransfection of Phoenix 293T cells with GFP+MSCV plasmids as previouslydescribed (Bowerman et 2011). HLA Class II proteins were expressed inhuman class II negative T2 cell line (T2 Parent). Below is describedclass II expression.

RNA was isolated from individuals expressing DRB1*03:01, DRB3*02:02,DQA1*05:01, DQB1*02:01, DQA1*03:01, DQB1*03:02, DRB1*15:01, DQA1*01:02,or DQB1*06:02, and complementary DNA (cDNA) for each individual HLA-DR,DQA1 or DQB1 allele was made. HLA-DRB1*04:01, DRB3*03:01, DRB4*01:03,and DRB5*01:01 T 2 cell lines were made previously (Anderson et al.2016). cDNA sequences for DRB1*11:03, DRB3*01:01, DQA1*05:05, andDQB1*03:01 were obtained from the IPD-IMGT/HLA Database (websiteebi.ac.uk/ipd/imgt/hla/) and were obtained as gBlocks from IntegratedDNA Technologies (Coralville, Iowa). cDNA was cloned into a murine stemcell virus (MSCV) plasmid for retroviral transduction of the human classII negative T2 cell line (T2 Parent). The HLA-DRB1, -DRB3, -DQA1, and-DQB1 alleles were individually packaged as retrovirus by transienttransfection of Phoenix 293T cells with GFP+MSCV plasmids as previouslydescribed (Bowerman et 2011). For the HLA-DRB1 and -DRB3 alleles, theretrovirus in the supernatant was used to transduce 1×10⁵ T2 cellsexpressing DRA1*01:01 and sorted for high expression of HLA-DR+/GFP+seven days post transduction (Anti-DR-APC (LN3) Invitrogen Cat#17-9956-42). For the -DQ alleles, the retrovirus of the HLA-DQB1alleles was used to transduce 1×10⁵ HLA Class II negative T2 cells andsorted for high GFP+ expression seven days post transduction. Then, theretrovirus for the corresponding HLA-DQA1 allele for the cis and transdimer was used to transduce 1×10⁵ DQB1+T2 cells and sorted for highHLA-DQ+/GFP+ expression seven days post transduction (Anti-HumanHLA-DP/DQ/DR Starbright Blue (WR18) 700 BioRad Cat #MCA477SBB700).Post-sort, RNA was isolated from each cell line to verify the HLAsequences for both cis and trans HLA alleles by Sanger sequencing(Quintara Biosciences). All cell lines were grown in IMDM-GlutaMAX (LifeTechnologies) supplemented with sodium pyruvate,thio-penicillin/streptomycin, and 10% fetal bovine serum (FBS).

Peptide Design and Synthesis for Peptide Binding Assays

Hybrid Insulin Peptides HIP1-WE14 (GQVELGGWSKMDQLA SEQ ID NO: 97),HIP6-IAPP2 (GQVELGGGNAVEVLK SEQ ID NO: 98), HIPS-NPY (GQVELGGGSSPETLISEQ ID NO: 99), and HIP11-C peptide (SLQPLALEAEDLQV SEQ ID NO: 100) weresynthesized with a biotinylated PEGS linker on the N-terminus to >98%purity with Trifluoroacetic acid (TFA) removal by Genscript (Piscataway,N.J.) (Delong 2016, Baker2019). The HIPs used in this study wereselected because of their capability to stimulate available T cellclones (Table 1). Biotinylated GAD65²⁶⁵⁻²⁸¹ (AMMIARFKMFPEVKEKG SEQ IDNO: 101), Insulin Mimotope (HLVEELYLVAGEEG SEQ ID NO: 102), andInfluenza A (PKYVKQNTLKLAT SEQ ID NO: 103) peptides were alsosynthesized as controls for HLA-DR and DQ binding [S. Dai, atdoi.org/10.1073/pnas.1716527115]. All peptides, except HIP6, werereconstituted in Dimethyl Sulfoxide (DMSO), then equal parts water, andfinally Dulbecco's phosphate buffered saline (DPBS) (Life Technologies)to 400 μM concentration and kept frozen at −20° C. until use in peptidebinding and T cell studies. HIP6 was reconstituted in 3% ammonia water,then equal parts water, 75 uL of 1M HCL to restore a neutral pH, andfinally DPBS to 400 μM.

Patient Specific HLA-Class II Expressing T2 Cells Peptide Binding

T2 cell lines expressing the HLA-Class -DR and -DQ genotype of Pt3977were harvested, resuspended, plated with 100 μM HIP1, and culturedovernight as mentioned above. Plates were washed twice with DPBS toremove unbound peptide, then resuspended in 100 μL 1:1000 dilutedeBioscience™ Fixable Viability Dye eFluor™780 for 30 min at 4° C. Then,the cells were processed and stained as before. Data were acquired onthe Canto II flow cytometer (BD Biosciences) and analyzed by FlowJoVersion X (Tree Star). The average binding ratio (MFI of HLA Class II+T2cells/MFI T2 parent HLA Class II -)±SEM for 3 independent experimentswas determined using GraphPad Prism software version 9.1.

Peptide Synthesis for T cell Stimulation Assays

Hybrid Insulin Peptides HIP1 (GQVELGGWSKMDQLA SEQ ID NO: 97), and HIP11(SLQPLALEAEDLQV SEQ ID NO: 100) were synthesized to >98% purity withTrifluoroacetic acid (TFA) removal by Genscript (Piscataway, N.J.)[Baker et al. 2019]. The peptides were reconstituted in DMSO to a finalconcentration of 10,000 μM. For the stimulation assays, the peptideswere diluted 1:100 for a working concentration of 100 μM.

Resistant and Susceptible HLA-Class II Expressing T2 Cells PeptideBinding

Peptide binding assays were conducted as described previously (Andersonet al. 2016, Roark et al. 2016). Briefly, T2 cell lines expressing T1Dresistant and susceptible HLA-DR and—DQ alleles were harvested andresuspended in media (IMDM-GlutaMAX, 10% FBS, thio-pen/strep and sodiumpyruvate) at 4×10⁶ cells/mL. In a 96-well round-bottom plate,resuspended cells, 100 μM biotinylated stock peptide, and DPBS werecombined. Negative control wells contained resuspended cells with DPBSalone. Plates were incubated overnight at 37° C. Plates were washedtwice with DPBS to remove unbound peptide, then resuspended in 1X ZombieAqua (Biolegend Zombie Aqua™ Fixable Viability Kit cat #423102) for 15min at room temperature. Cells were lightly fixed for five minutes in 1%formaldehyde in DPBS to prevent loss of peptide from the cell surface.To detect peptide binding, 1X PE-labeled streptavidin (One LambdaLT-SA-PE) was added for 30 min at 4° C. Prior to acquisition on theCanto II flow cytometer (BD Biosciences), cells were again fixed. Datawere analyzed by FlowJo Version X (Tree Star) and the average bindingratio (MFI of HLA Class II+T2 cells/MFI T2 parent HLA Class II -)±SEMfor 3 independent experiments was determined using GraphPad Prismsoftware version 9.1 (Graph Pad).

For the titration of HIP11, the T2 Parent, HLA-DQ2, and—DQ2 trans wereharvested and resuspended as mentioned above. Then, in a 96-well roundbottom plate, the reaction was setup as before except the finalconcentrations of peptide were 5 μM, 10 μM, 20 μM, and 50 μM. The cellswere cultured overnight, and washed twice with DPBS. Cells wereresuspended in 100 μL 1:1000 diluted eBioscience™ Fixable Viability DyeeFluor™780 (cat #65-0865-18) for 30 min at 4° C. Then, the cells wereprocessed, stained, and analyzed as mentioned above.

T cell Stimulation Assay

T cells were cloned and expanded as described previously (Baker 2019).For HIP11, HLA-DQ2 and -DQ2 trans expressing T2 lines or autologousEBV-transformed B-cell line (EBV3537) were either unloaded or preloadedwith varying concentrations of HIP11 (5 μM, 10 μM, 20 μM, and 50 μM).The antigen presenting cells were preloaded by incubating the antigen atthe selected concentrations with the cells for 1 hr at 37° C. Then,excess antigen was removed by washing with DPBS to ensure only theantigen bound and presented by the HLA alleles was capable ofstimulating the T cell clones. Then, 1×10⁵ CD4+ T cell clones (E2) wereincubated with 5×10⁴ of the antigen presenting cell lines overnight thenstained with viability dye (eBioscience™ Fixable Viability DyeeFluor™780) for 30 min at 4° C. The cells were washed then stained withanit-CD4-PE (Biolegend PE anti-human CD4 Antibody cat #317410), andanti-CD25-BV421 (BD Biosciences BV421 Mouse Anti-Human CD25 cat #562443)for 30 min at 4° C. Cells were washed then fixed before acquisition onthe Canto II flow cytometer (BD Biosciences). Data were analyzed byFlowJo Version X (Tree Star). GraphPad Prism software version 9.1 wasused to calculate the mean CD25 MFI±SEM of 3 independent experiments.

For HIP1, 1×10⁵ CD4+ T cell clones (D11) were incubated with 5×10⁴patient specific HLA-Class II T2 lines or autologous EBV-transformedB-cell line (EBV 3977) in the absence or presence of antigen. TheHLA-Class II T2 cell lines and EBV line were preloaded as mentionedabove with a concentration of 20 μM. The cells were co-culturedovernight, and they were processed and analyzed as mentioned above.

Example 2—Humanized DRB1*04:01^(K71E) Transgenic Mice are Resistant toCollagen Sensitization

Collagen-induced arthritis (CIA) is a well-established mouse model ofautoimmune arthritis that recapitulates key features of RA, including animportant role of MHC II molecules and collagen specific T cellresponses. HLA-DR4 transgenic mice injected with heterologous type IIcollagen protein emulsified in Complete Freund's Adjuvant (CFA) developpotent collagen-specific CD4+ T cell responses (collagen sensitization),an essential first step required for development of CIA.

In order to determine if a DRB1*04:01-K71E gene edit is sufficient toprevent collagen sensitization in vivo, chimeric HLA-DR4/I-E^(d)transgenic mice on an H-2 class II knockout background were used. FIG. 2, top, shows a diagram showing the distal/human DRα1 and DRβ1 domains inthe chimeric MHC II molecules mediate peptide binding and interactionwith the mouse T cell receptor (mTCR), while the proximal murineI-E^(d)α2 and I-E^(d)α2 domains mediate interactions with the mouse CD4co-stimulatory molecule. Three transgenic lines were used in theseexperiments: one carrying the DRB1*04:01 gene one carrying theDRB1*01:01 gene (see, for example (J. Exp. Med., Vol. 180, 1994, pp.173-18, and J. of Exp. Med., Vol. 185, No. 6, 1997, p. 1113-1122, bothof which are incorporated by reference in their entireties), and onecarrying the DRB1*04:01^(K71E) gene (based on the methods in PLOS ONE,Vol. 8, 12, 2013, e84908, which is incorporated by reference). All threelines were immunized on Day 0 and Day 21 with soluble type II collagenprotein emulsified in CFA. On Day 56, mice were sacrificed, lymph nodeswere harvested, and cultured with collagen²⁵⁸⁻²⁷² peptide (Pep) or inmedia alone (No Pep) in the presence of the thymidine nucleoside analog5-ethynyl-2′-deoxyuridine (EdU) that incorporates into the DNA ofproliferating cells.

Fluorescent azide and Cu(I)-catalyzed [3+2] cycloaddition “click”chemistry was used to detect EdU incorporation in proliferating cells.Cells were also co-stained with fluorescent antibodies for CD3 and CD4.The frequency of CD4+ T cells that proliferated ex-vivo in response tocollagen²⁵⁸⁻²⁷² was used to quantify sensitization. As shown in FIG. 2 ,bottom, DRB1*04:01 mice developed collagen²⁵⁸⁻²⁷² specific CD4+ T cellresponses, while CD4+ T cells from DRB1*01:01 transgenic mice exhibiteda weak proliferative response, commensurate with its reduced ability tobind collagen²⁵⁸⁻²⁷² and weaker association with RA compared toDRB1*04:01. In contrast, there was no proliferative response in CD4+ Tcells from DRB1*04:01^(K71E) transgenic mice. This demonstrated thatexpression of DRB1*04:01^(K71E) in otherwise sensitive mice preventedthe mice from becoming sensitized to collagen.

Example 3—DRB1*04:01^(K71E) Skin Transplants Achieve Stable Engraftmentin DRB1*04:01 Mice

To verify that the K71E edit would not induce alloreactivity inDRB1*04:01 recipients, skin transplants were performed from eitherDRB1*01:01 and DRB1*04:01^(K71E) mice onto DRB1*04:01 recipients. Skingrafts contain an abundance of APCs making it a difficult tissue toengraft. Assessment of skin engraftment is a robust model to test forpotential alloreactivity. This pre-clinical model was used to determinethe frequency of DRB1*04:01^(K71E) skin graft rejection in DRB1*04:01recipients.

As shown in FIG. 3 , bottom, DRB1*01:01 skin grafts were completelyrejected by day 14 (n=3), but the DRB1*04:01 (n=3) and DRB1*04:01^(K71E)(n=3) grafts both remained stably engrafted. The fact that theDRB1*04:01^(K71E) allografts survived indefinitely indicates thatDRB1*04:01^(K71E) expression does not induce either acute or chronicrejection. Therefore, once engrafted into the bone marrow, long-termprogenitor HSCs expressing DRB1*04:01^(K71E) should not be rejected orinduce an immune response in the DRB1*04:01 recipient.

Example 4—HLA Alleles Involved in MS

Alleles associated with MS resistance and susceptibility wereinvestigated. Two DRB1 alleles, *01:01 and *11:01 were identified asalleles conferring resistance, while *15:01 is associated withsusceptibility. The mature protein sequence of the three alleles wassubmitted to alignment, and polymorphic positions matching the criteriadescribed above were identified for the DRB1*15:01 allele: F47, A71, andV86. Mutations were made at one or more of these positions (F47Y, A71R,and V86G) against 4 peptides: MOG⁹⁷⁻¹⁰⁹ (FFRDHSYQEEA SEQ ID NO: 121),which is related to MOGAD; RASGRP²⁷⁸⁻⁸⁷ (LVRYWISAFP SEQ ID NO: 122),which is expressed in the brain and may activate memory T cells in MS,leading to characteristic brain inflammation (Jelcic et al., 2018, Cell175); MBP⁸³⁻¹⁰¹ (ENPVVHFFKNIVTPRTPPP SEQ ID NO: 123), an immunogenicpeptide that binds DRB1*15:01; and MBP¹⁴⁶⁻¹⁷⁰) AQGTLSKIFKLGGRDSRSGSPMARRSEQ ID NO: 124), which may play a role in resistance to MS, it bindsDRB1*01:01 and does not activate MS T cells (Mamedov et al., Front.Immunol. 2020.)

The collected data (FIGS. 5-7 ) indicate that DRB1*15:01, which conferssusceptibility to MS, binds the RASGRP2 better than the two resistantalleles—it also binds the MBP⁸³⁻¹⁰¹ peptide strongly, but not theMBP¹¹⁴⁶⁻¹⁷⁰ peptide. These data also suggest that the V86G and F47Ymutations affected the binding pattern mildly, except with theMBP¹⁴⁶⁻¹⁷⁰ peptide, where binding is better, suggesting that thispeptide may play a role in resistance. Finally, The A71R mutation alonein DRB1*15:01 does not change peptide binding except to increase thebinding of MBP 146-170 (FIG. 6 ). The double mutation, A71R-V86G, showsa decrease in RASDRP2 binding as well (FIG. 7 ), suggesting that doublemutations may provide additional benefits in addressing MS autoimmunity.

Example 5—HLA Alleles Associated with NMO (Neuromyelitis Optica)

The clinical syndrome of NMO is characterized by acute optic neuritisand transverse myelitis, caused by pathogenic serum IgG autoantibodiesto aquaporin 4 (AQP4). AQP4 is the most abundant water-channel proteinin the central nervous system (>80% of cases). Susceptibility to NMO isassociated with HLA-DRB1*03:01, while resistance is associated with theallele DRB1*07:01. Two AQP4 peptides were tested for their binding toboth alleles.

The collected data indicate that the AQP4-5 peptide binds to thesusceptible allele and not the resistant allele (FIG. 9 ). The same isseen with the AQP4-6 peptide, which also binds the susceptible allele,but not the resistant allele. These results suggest that the peptidebinding profile of DRB1*03:01 may be affected by mutating positions inthe peptide binding groove, to prevent or lessen binding of AQP4peptides, and thus prevent autoimmunity. Candidate positions (e.g. asdisclosed elsewhere target amino acid positions 9, 11, 13, 26, 28, 30,32, 33, 37, 38, 40, 47, 57, 58, 67, 71, 74, 78, 85, and 86) for such amutation can be identified from the sequence alignment of DRB1*03:01 andDRB1*07:01 (FIG. 8 ; note positions 38, 40, and 85 are not polymorphicindicate 03:01 and 07:01; note also that aligned sequence positions arenumbered based on mature protein sequence).

Example 6—HLA Alleles Associated with RA

The HLA-DRB1 allele *04:05 also shows susceptibility to RA. Inparticular, this allele shows a strong association with RA in Japanesepopulation. For these studies, position 71 in DRB1*04:05 was mutatedfrom R to E (see FIG. 10 ).

These studies indicate that DRB1 allele *04:05 does not show a strongpreference for binding the immunodominant collagen peptide (FIG. 11 ).However, when position 71 is mutated to glutamic acid (R71E), the lowlevel of binding is further reduced. In the case of vimentin andα-enolase, the *04:05 allele preferentially binds the citrullinatedversions over the native versions. As with to the K71E mutation, thispreference is reduced by changing arginine at position 71 in DRB1*04:05to glutamic acid (R71E). MFI Binding Ratio averages for 2 experiments.

As disclosed herein, other DR4 alleles susceptible to RA, for exampleDRB1*04:03, *04:04, and *04:08, may confer resistance (i.e. resistantalleles) when position 71 is changed from arginine (R) to glutamic acid(E).

Example 6—HLA Alleles Associated with Type I Diabetes— T1D

DQB1 alleles associated with susceptibility to Type I diabetes weretested for the ability to confer resistance with one or more mutationsin the antigen binding groove. Specifically, several DQB alleles andcorresponding A57D variants were cloned into T2 cell lines. Asparticacid is found at position 57 in several resistant alleles.

TABLE 1 Selected Hybrid Insulin Peptide Information (See also FIG. 22)Hybrid SEQ Insulin Abbrevi- ID Peptides ations Sequences References NOHIPI-WE14 HIP1 GQVELGG- Delong et al. 2016 97 WSKMDQLA HIP6-IAPP2 HIP6GQVELGGG- Delong et al. 2016 98 NAVEVLK HIP8-NPY HIP9 GQVELGGG-Delong et al. 2016 99 SSPETLI HIP11-C HIP11 SLQPLAL- Baker et al. 2019100 EAEDLQV

Peptide Selection -Hybrid Insulin Peptides HIP1-WE14 (GQVELGGWSKMDQLASEQ ID NO: 97), HIP6-IAPP2 (GQVELGGGNAVEVLK SEQ ID NO: 98), HIPS-NPY(GQVELGGGSSPETLI SEQ ID NO: 99), and HIP11-C peptide (SLQPLALEAEDLQV SEQID NO: 100) were synthesized with a biotinylated PEGS linker on theN-terminus to >98% purity with Trifluoroacetic acid (TFA) removal byGenscript (Piscataway, N.J.) (Delong 2016, Baker2019). The HIPs used inthis study were selected because of their capability to stimulate andavailability of T cell clones (Table 1). Biotinylated GAD65²⁶⁵⁻²⁸¹(AMMIARFKMFPEVKEKG SEQ ID NO: 101), Insulin Mimotope (HLVEELYLVAGEEG SEQID NO: 102), and Influenza A (PKYVKQNTLKLAT SEQ ID NO: 103) peptideswere also synthesized as controls for HLA-DR and DQ binding [S. Dai,available at doi.org/10.1073/pnas.1716527115].

Hybrid insulin peptides were tested for their binding to these celllines at various concentrations. Specifically, peptide binding of thenative HLA DQB1 allele was compared with its A57D mutated form.Susceptible alleles were hypothesized to bind the hybrid insulinpeptides.

These studies showed that the susceptible DQ2 and DQ8 alleles do notbind the HIP8-NPY peptide, but the DQ2 trans HLA molecule does (FIG. 12). When position 57 is changed from A to D, the binding of this hybridinsulin peptide is increased on DQ2 and DQ8 but is reduced on DQ2 trans.Similarly, DQ2 and DQ8 do not bind the HIP11-C peptide (FIG. 13 ), butthe DQ2 trans molecule does. When A57D is introduced, DQ2 shows peptidebinding. While DQ2 trans binds this peptide less, binding is notabolished. Binding of the insulin mimotope peptide follows a similarpattern as to the above peptides (FIG. 14 ). Specifically, DQ2 does notbind the mimotope peptide, but DQ2 trans and DQ8 bind the peptide. Whenthe A57D mutation is introduced, DQ2 and DQ2 trans now bind the peptide.While the mutation reduces binding to the DQ8 molecule.

The effect on T cell stimulation by the A57D mutation was also tested.Specifically, E2 T-cells, restricted to DQ2 and specific for the HIP11-Cpeptide, were obtained. These T cells were stimulated in culture with T2cells expressing DQ2 or the DQ2 trans molecule in the presence ofdifferent concentrations of the HIP11-C peptide overnight. Bothmolecules with the A57D mutations, were also tested. T cell stimulationwas then measured by staining the cells for the IL-2R (CD25) on thesurface of the cells.

These studies showed that the DQ2 T2 cell lines stimulate the E2 T cellclone much better than the parent EBV line (FIG. 15 , top panel).Introducing the A57D mutation into these alleles results in lessstimulation of the E2 T cell. As shown in the bottom panel of FIG. 15 ,the E2 T cell clone is stimulated by the DQ2 trans molecule but theintroduction of A57D into the DQ2 trans molecule results in lessstimulation of the T cell clone.

FIG. 16 shows HLA-DQ alleles binding Hybrid Insulin Peptides. Binding ofbiotinylated HIP1-WE14, HIP6-IAPP2, HIP9-NPY, and HIP11-C peptide weremeasured on T2 cells expressing the risk alleles of DQ2(A1*05:01/B1*02:01), DQ8 (A1*03:01/B1*03:02), DQ2 trans(A1*03:01/B1*02:01), and DQ8 trans (A1*05:01/B1*03:02). The resistantallele of DQ6 (A1*01:02/B1*06:02) was also tested. The light gray is thebackground binding of the peptide to the HLA-Class II (−) T2 Parent linewhile the darker gray is peptide binding to the specific HLA-Class II(+) T2 line. The rows represent different alleles, and the columns aredifferent peptides. The number in the upper right corner is the averagebinding ratio (SA-PE MFI T2 HLA Class (+)/SA-PE MFI T2 parent). Thenumber represents the average binding ratio from 3 independentexperiments.

FIG. 17 shows HLA-DQ alleles binding control native peptides. Binding ofbiotinylated Insulin Mimotope and GAD65²⁶⁵⁻²⁸¹ were measured on T2 cellsexpressing the risk alleles of DQ2 (A1*05:01/B1*02:01), DQ8(A1*03:01/B1*03:02), DQ2 trans (A1*03:01/B1*02:01), and DQ8 trans(A1*05:01/B1*03:02). The resistant allele of DQ6 (A1*01:02/B1*06:02) wasalso tested. The light gray is the background binding of the peptide tothe HLA-Class II (−) T2 Parent while the darker gray is signal of theHLA-Class II (+) T2 line. The number in the upper right corner is theaverage binding ratio (SA-PE MFI T2 HLA Class (+)/SA-PE MFI T2 parent).The number represents the average binding ratio from 3 independentexperiments.

FIG. 18 shows susceptible and resistant HLA-DRB1 alleles binding HIPs.Binding of biotinylated HIP1, HIP6, HIP8, and HIP11 were measured on T2cells expressing the susceptible alleles of DRB1*03:01 and DRB1*04:01and resistant DRB1*15:01. The light gray is the background binding ofthe peptide to the HLA-Class II (−) T2 Parent line while the darker grayis signal of the HLA-Class II (+) T2 line. The number in the corner isthe mean binding ratio of 3 independent experiments.

FIG. 19 shows susceptible and resistant HLA-DRB1 alleles binding nativecontrol peptides. Binding of biotinylated insulin mimotope,GAD65²⁶⁵⁻²⁸¹, and Influenza HA were measured on T2 cells expressingeither susceptible or resistant HLA-DRB1 alleles, specificallyDRB1*03:01, DRB1*04:01, and HLA*DRB1*15:01. The number in the corner isthe mean binding ratio.

FIG. 20 shows various HLA-DRB3/4/5 alleles binding HIPs. The ability ofthe HLA-DRB3/4/5 alleles to bind biotinylated HIP1, HIP6, HIP8, andHIP11 were measured on T2 cells expressing the alleles of DRB3 (*01:01,*02:02, *03:01), DRB4*01:03, and DRB5*01:01. The light gray is thebackground binding of the peptide to the HLA-Class II (−) T2 Parent linewhile the darker gray is signal of the HLA-Class II (+) T2 line. Thenumber in the corner is the mean binding ratio of 3 independentexperiments.

FIG. 21 shows various HLA-DRB3/4/5 alleles binding native controlpeptides. Binding of biotinylated insulin mimotope, GAD65²⁶⁵⁻²⁸¹, andInfluenza HA were measured on T2 cells expressing DRB3 (*01:01, *02:02,*03:01), DRB4*01:03, and DRB5*01:01. The light gray is the backgroundbinding of the peptide to the HLA-Class II (−) T2 Parent line while thedarker gray is signal of the HLA-Class II (+) T2 line. The number in thecorner is the mean binding ratio of 3 independent experiments.

Example 7—Effect of Mutations in Pocket 1 of DRB1

Disclosed herein are methods and compositions for occluding the antigenbinding position, pocket 1, of an HLA class II protein. In manyembodiments, the HLA class II protein is DRB, for example DRB1, DRB3,DRB4, or DRB5. In many embodiments, variant DRB molecules are describedwhere one or more amino acid positions in pocket 1 are edited. In manyembodiments, the edit may include changing a glycine to a larger aminoacid identity, for example valine, methionine, or leucine. In manyembodiments, the amino acid position is 85 or 86 in the mature DRBprotein.

Position 86 is located within pocket 1 of the peptide binding region ofDRB1, and may act as a peptide anchor position deep in the bindinggroove. Pocket 1 (or P1) of DRB molecules may form a deep recess,depression, or hole for receiving one or more large amino acid sidechains to aid in anchoring the peptide antigen. Studies were designed toinvestigate the effect of replacing glycine at position 86 of DRB1 withother amino acids. Specifically, larger, non-polar amino acids, valine,methionine, and leucine, were substituted to reduce the size of thispeptide anchor position.

In these studies, a specific amino acid position within the bindingcleft of DRB1*04:01, position 86, was changed from glycine to eithermethionine (G86M) or leucine (G86L). T2 cell lines were transfected withengineered genes coding for the variants and the novel HLA moleculeswere expressed. Cell lines were incubated overnight with 100 uMbiotinylated peptide. The next day, cells were washed and lightly fixedbefore the addition of streptavidin-PE to detect bound biotinylatedpeptide. Cells were analyzed by flow cytometry using the BD Cantoinstrument. A variety of peptides were tested to see how changing pocket1 might affect peptide binding: increase, decrease or remain the same.

T2 cell lines expressing either DRB1*04:01 or DRB1*04:01 where position86 was changed from glycine to methionine (G86M) or glycine to leucine(G86L) were tested for their ability to bind a variety of peptides.Position 86 is located in pocket 1 of the peptide binding region of DRB1and we hypothesized that replacing glycine with a larger, non-polaramino acid, we could essentially block peptide binding by filling upthis anchor position.

DRB1*04:01 binds all peptides listed here (FIG. 23 ). When position 86is changed to a methionine or leucine, inhibition of peptide binding isobserved for collagen, MOG, and GAD65. A reduction in peptide binding isseen for the insulin mimotope and the viral influenza HA peptide. Thelight grey peak is binding by the T2 parent cell line A. The dark peakis peptide binding by the specific HLA allele. Number in the right-handcorner is the MFI ratio over background binding to the T2 parent linethat does not express class II HLA. Bold number is the binding ratio fortwo experiments for HA, collagen, MOG, and GAD65; Insulin Mimotope onlyhas one experiment.

The same cell lines were tested with hybrid insulin peptides (FIG. 24 ).Changing position 86 from G to M or G to L did not result in a gain offunction for these hybrid insulin peptides. They do not bind toDRB1*04:01 or the two mutants.

As shown in FIG. 25 , binding of RASGRP2 peptide, which is thought toplay a role in MS, was reduced on the two mutants. However, the mutantcell lines still bound MBP peptides. The AQP4 peptides (NMO) boundsimilarly to DRB1*04:01 and the two mutants.

There is a preference for binding citrullinated vimentin andcitrullinated α-enolase peptides are reduced when position 86 is changedto M or L. FIG. 26 depicts these binding with the α-enolase peptides.These mutations do not block all peptide binding. Light gray iscitrullinated, dark gray is native peptide. The very light grey peak isbackground binding of the peptide to a T2 cell that does not express theHLA molecule. Ratio of citrullinated to native peptide binding is shownin the right-hand corner.

Arthritogenic peptides were tested against DRB1*04:01 and the twomutants. FIG. 27 shows comparisons of the native versus citrullinatedversions of the peptides on these cell lines. These studies show thatthe native and citrullinated versions of vimentin bind better whenposition 86 is mutated. Native α-enolase binding is increased on theG86L cell line but both mutants bind less of the citrullinated from.Here the ratio is calculated over the T2 parent background.

Example 8— Bone Marrow Treatments

At least 48 hrs before the experiment, mice were fed Baytril water(Baytril at 22.7 mg/ml: 50 ml tube with 1135 mg enrofloxacin, 45 mlwater, 1.25 ml 1-butanol (n-butanol) and add dropwise (50% NaOH, 19M)45% KOH (11.7M) until enrofloxacin dissolves (˜150-200 μI); check pH,adjust to pH 8.9-10.9 with HCl, and qs to 50 ml with water) at 2 ml ofBaytril water per water bottle (˜375 ml). Bone marrow cells wereisolated from the donor K71E mice and transferred into irradiated DR4recipients. Recipient mice were irradiated with 2 doses @ 6 hrs apart toachieve total dose.

Bone Marrow Reconstitution

Bone marrow cells were isolated from bones after washing in ethanol anddissecting away all the muscle tissue and tendons. Ends of the bone werecut and flushed using a 10 ml syringe with a 25-gauge needle to removebone marrow cells. Cells were flushed into a 10 ml tissue culture platewith fresh PBS. Bone marrow cells were disrupted by passaging the cellsthrough an 18-gauge needle/10 mL syringe with the PBS in the plate.Single cell suspension was transferred to a 50 ml conical tube, whichwas centrifuged to pellet cells. Red blood cells were lysed byincubating 1 min with 2 ml RBC lysis buffer, and then composition wasfiltered through a 70 uM filter into a fresh 50 ml tube. 10 ul aliquotwas diluted 1:2 in trypan blue for staining. Non-RBC cells were countedand total number of cells for each type of donor tallied. Cells wereagain centrifuged at 400×g and resuspend in PBS to 2−5×10⁷ cells/mL. 100ul was transferred to each irradiated recipient mouse by retro-orbitalinjection.

Monitoring and Checking for Reconstitution and Chimerism

Mice were monitored daily for the first 2 weeks and thereafter on aweekly basis. Baytril water was replaced once a week for 4 weeks. Bloodsamples from mice were checked for reconstitution, by staining for B andT cells after 6 weeks. Mice with donor cell markers were checked forchimerism using flow cytometry. If no cell surface marker is available,samples were submitted for genotyping to test for the presence ofaltered gene or gene disruption.

Bone Marrow Transplant Using Anti-CD117 or Other Reagents to RemoveCells in the Recipient for the Transplant.

Anti-CD117 antibodies may aid engraftment of the disclosed engineeredHSCs because they target HSCs (2019 May 9; 133(19):2069-2078 doi:10.1182/blood-2018-06-858159). Anti-human CD117 mAb, SR-1, inhibitsnormal cord blood and bone marrow HSCs in vitro. SR-1 and clinical-gradehumanized anti-human CD117 mAb, AMG 191, deplete normal and MDS HSCs invivo in xenograft mouse models. These anti-CD117 mAbs are also useful infacilitating engraftment of normal donor human HSCs in MDS xenograftmouse models, restoring normal human hematopoiesis and eradicatingaggressive pathologic MDS cells, in some cases the anti-CD117 antibodyhelps to block binding of hematopoietic stem cells to the bone marrowstroma, thus releasing them from the bone marrow into the peripheralcirculation. For this reason, one method of treating a subject having orat risk of developing an autoimmune disease may include prior treatmentwith an anti-CD1117 antibody to aid engraftment of engineered HSCscomprising the disclosed variant HLA molecules. Alternatively, subjectsmay be subjected to mobilization of immune cells with GCF treatments,prior to administration of engineered cells, as disclosed for harvestingof HSCs above.

Mice are placed into two groups. For these experiments, Group I wereDR4+ mice, which were given two retrorbital iv injections of anti-CD117(day 0 and day 2). These mice then received bone marrow cells fromDRB1*04:01K71E donors on day 8. There after blood from recipients wascollected at two time points (day 14 and day 28 after BMT) and analyzed.Group II mice were also DR4+, but received no anti-CD117 (day 0 and day2). However, they did receive K71E bone marrow cells on day 8.Thereafter, blood was collected at two time points (day 14 and day 28after BMT) as for Group I. Final samples collected on day 56

Blood samples are analyzed using digital PCR to look for the singleamino acid difference between DR4 mice and K71E mice.

Busulfan, an alkylating chemotherapeutic agent, may also be used toprepare subjects for receipt of allogenic engineered HSCs. In someembodiments, busulfan is also used to treat recipient mice prior totransfer of bone marrow cells from donor mice carrying the engineeredDRB1*04:01K71E allele.

Discussion

The experiments and data disclosed herein provide original proof ofconcept for treatment of autoimmune diseases, including RA as well asType 1 diabetes, multiple sclerosis, neuromyelitis optica, and otherdisorders arising from undesirable HLA protein-mediated binding andpresentation of self-peptides to immune effector cells. The presentdisclosure is also to Applicant's knowledge the first description oftreatment of an autoimmune disease other than RA. As such, the presentdisclosure is to Applicant's knowledge the first to broadly enable anddemonstrate possession of treatment of autoimmunity by HLA engineeringas disclosed.

The present disclosure further provides the novel HLA engineeringstrategy of steric occlusion of the antigen binding pocket (Pocket 1) ofan HLA class II protein to modify binding and presentation of peptides,including self-peptides recognized as antigenic in autoimmune disease.In particular, the present disclosure describes and exemplifies (seeExample 7) the strategy of replacing a relatively small amino acid(e.g., glycine) with a relatively large amino acid (e.g., methionine) togenerally reduce the amount and affinity of peptide binding by an HLAprotein associated with autoimmunity.

Thus, as broadly embodied, the present disclosure provides, inter alia,methods of treating or preventing autoimmunity by HLA engineering andmethods of designing an HLA engineering treatment for autoimmunedisease. The HLA engineering, which can be performed in vivo or ex vivo,reduces binding of one or more self-peptides associated with autoimmuneresponse, and is designed and conducted to replace one or more aminoacids that contribute to binding of that self-peptide(s) by the HLAprotein, wherein the one or more amino acids are relatively‘immunoprivileged’ by virtue of their location(s) within the HLAprotein's antigen binding cleft.

The substituted or replacement amino acid can be identified by referenceto an HLA allele associated with resistance to autoimmunity, such as aparticular autoimmune disease by which a subject is afflicted or towhich the subject is considered vulnerable. In certain embodiments, forexample, candidate HLA protein amino acid residues for engineering areidentified by comparison of the sequences and/or three-dimensionalmodels of the autoimmune disease-associated HLA protein and an HLAprotein associated with resistance to the same autoimmune disease. Suchthree-dimensional models include crystal structures of the HLA proteinsin complex with a peptide or peptides associated with the autoimmunedisease. Additionally or alternatively, the replacement amino acid canbe identified de novo, such as by in silico modeling and/orhigh-throughput in vitro assays to identify substitutions that reducebinding of the HLA protein to the autoimmunity-associated peptides(e.g., peptides derived from insulin, collagen, RASDRP2, in diabetes,RA, and MS, respectively).

In some embodiments, the methods comprise identifying a small aminoacid, such as glycine, at a suitable location, such as Pocket 1, of anHLA protein associated with an autoimmune disease, and engineering thecorresponding HLA allele to express a replacement amino acid that islarger in size. The methods can further include assaying binding ofself- and/or nonself peptides to the engineered HLA protein, as well asthe functional assaying discussed above.

As contemplated herein, and as expressly described at, for example,Example 4, the HLA engineering can include replacement (or mutation) oftwo or more amino acids in an HLA protein, and the methods of designingtreatment can be applied and optimized accordingly.

Certain embodiments provide methods of treating or preventing anautoimmune disease and methods of designing a treatment for autoimmunedisease by HLA engineering. Certain embodiments provide methods oftreating or preventing RA, T1D, MS, neuromyelitis optica, Behget'ssyndrome, celiac disease, and psoriasis, and methods of designing atreatment for same. Certain embodiments provide methods of treating orpreventing T1 D, MS, neuromyelitis optica, Behget's syndrome, celiacdisease, and psoriasis, and methods of designing a treatment for same.In certain embodiments, the HLA engineering does not compriseDRB1*04:01″E mutation. In certain embodiments, the HLA engineering doesnot comprise mutation of position 71 of the DRB1*04:01 allele. Incertain embodiments, the HLA engineering does not comprise mutation ofthe DRB1*04:01 allele.

Significantly, many embodiments of the present disclosure, includingcertain embodiments, do not require, and can exclude, post-treatmentimmunosuppression. Accordingly, certain methods of designing a treatmentfor autoimmune disease by HLA engineering according to the presentdisclosure comprise, for example, in vitro T cell stimulation assaysand/or skin graft experiments to confirm efficacy and non-rejection ofcandidate mutations, wherein such efficacy and/or non-rejectionidentifies a suitable mutation for HLA engineering as disclosed herein.

While multiple embodiments are disclosed, still other embodiments of thepresently disclosed concepts, compounds, compositions, methods,processes, systems, and therapies will become apparent to those skilledin the art from the following detailed description. As will be apparent,the present disclosure is capable of modifications in various obviousaspects, all without departing from the spirit and scope of the presentdisclosure. Accordingly, the detailed description is to be regarded asillustrative in nature and not restrictive.

All references disclosed herein, whether patent or non-patent, arehereby incorporated by reference as if each was included at itscitation, in its entirety. In case of conflict between reference andspecification, the present specification, including definitions, willcontrol.

Although the present disclosure has been described with a certain degreeof particularity, it is understood the disclosure has been made by wayof example, and changes in detail or structure may be made withoutdeparting from the spirit of the disclosure as defined in the appendedclaims.

What is claimed is:
 1. A method of modifying an HLA allele associatedwith an autoimmune disease, comprising: identifying an HLA geneassociated with an autoimmune disease; identifying a susceptible HLAallele of the identified HLA gene, wherein the susceptible HLA allele isassociated with susceptibility to the autoimmune disease; identifying aresistant HLA allele of the identified HLA gene, wherein the resistantHLA allele is associated with resistance to the autoimmune disease;identifying one or more target amino acid positions within a bindingcleft of an HLA protein encoded for by the susceptible HLA allele,wherein the target amino acid position has a first identity and thetarget amino position in the HLA protein encoded by the resistant HLAallele has a different, second identity; modifying the susceptible HLAallele to encode an HLA protein with the target amino acid positionhaving the second identity and with altered binding affinity for atleast one self-peptide as compared to binding affinity of a HLA proteinencoded for by the susceptible HLA allele for the at least oneself-peptide; and thereby modifying an HLA allele associated with anautoimmune disease.
 2. The method of claim 1, wherein the autoimmunedisease is selected from rheumatoid arthritis (RA), celiac disease,diabetes mellitus type 1, systemic lupus erythematosus (SLE), multiplesclerosis (MS), myelin oligodendrocyte glycoprotein antibody disorders(MOGAD), myasthenic syndromes and neuromyelitis optica (NMO), ankylosingspondylitis, Behget's syndrome, Birdshot uveitis, narcolepsy, narcolepsytype 1 (NT1; previously termed narcolepsy with cataplexy), Kawasakidisease, Crohn's disease, psoriasis, dermatomyositis (DM), Addison'sdisease, irritable-bowl syndrome (IBS), Graves' disease,Henoch-Schönlein purpura (HSP), sarcoidosis, Sjögren's syndrome,eosinophilic granulomatosis with polyangiitis, Hashimoto's disease,idiopathic thrombocytopenic purpura, polymyositis (PM), paraneoplasticneurological syndromes (PNS), autoimmune encephalitis, lupus nephritis(LN), myasthenia gravis (MG), psoriatic arthritis, graft rejection,graft-versus-host disease (GVHD), an unwanted delayed-typehypersensitivity reaction, T-cell mediated pulmonary disease, neuritis,vitiligo, autoimmune pancreatitis, inflammatory bowel diseases,ulcerative colitis, glomerulonephritis, scleroderma, autoimmune thyroiddiseases, asthma, autoimmune uveoretinitis, pemphigus vulgaris,pulmonary fibrosis or idiopathic pulmonary fibrosis, primary biliarycirrhosis, and pernicious anemia.
 3. The method of claim 2, wherein thetarget amino acid is not at the T-cell receptor binding interface. 4.The method of claim 3, wherein the self-peptide contains at least onedeiminated residue.
 5. The method of claim 3, wherein the HLA gene codesfor a Class I or Class II HLA protein.
 6. The method of claim 5, whereinthe HLA gene codes for HLA-A, HLA-B, or HLA-C.
 7. The method of claim 6,wherein the susceptible HLA allele is selected from A*02, A*03, andA*29.
 8. The method of claim 7, wherein the susceptible HLA allele isselected from A*02:01, A*03:01, and A*29:01.
 9. The method of claim 6,wherein the susceptible HLA allele is selected from B*07, B*08, B*27,B*51, B*54, and B*57.
 10. The method of claim 9, wherein the susceptibleHLA allele is selected from B*07:02, B*08:01, B*27:03 B*27:05, B*27:09,B*51:01, B*54:01, and B*57:01.
 11. The method of claim 10, wherein thetarget amino acid position is position 59 or position 116, or both, andthe second identity is histidine.
 12. The method of claim 6, wherein thesusceptible HLA allele is selected from C*06 and C*18.
 13. The method ofclaim 12, wherein the susceptible HLA allele is selected from C*06:02and C*18:01.
 14. The method of claim 5, wherein the HLA gene codes forone of HLA-DPA, HLA-DPB, HLA-DQA, HLA-DQB, HLA-DRA, AND HLA-DRB.
 15. Themethod of claim 14, wherein the susceptible HLA allele is DPA*02. 16.The method of claim 15, wherein the susceptible HLA allele is DPA*02:01.17. The method of claim 14, wherein the susceptible HLA allele isDPB*13.
 18. The method of claim 17, wherein the susceptible HLA alleleis DPB*13:01.
 19. The method of claim 14, wherein the susceptible HLAallele is selected from DQA1*01, DQA1*02, DQA1*03, and DQA1*05.
 20. Themethod of claim 19, wherein the susceptible HLA allele is selected fromDQA1*01:01, DQA1*01:02, DQA1*02:01, DQA1*03:01, DQA1*05:01 andDQA1*05:05.
 21. The method of claim 14, wherein the susceptible HLAallele is selected from DQB1*02, DQB1*03, DQB1*05, and DQB1*06.
 22. Themethod of claim 21, wherein the susceptible HLA allele is selected fromDQB1*02:01, DQB1*03:01, DQB1*03:02, DQB1*05:01, DQB1*06:01 andDQB1*06:02.
 23. The method of claim 22, wherein the target amino acidposition is position 57 or position 71, or both, and the second identityis selected from aspartic acid, glutamic acid, and tyrosine.
 24. Themethod of claim 14, wherein the susceptible HLA allele is selected fromDRB1*01, DRB1*03, DRB1*04, DRB1*07, DRB1*08, DRB1*09, DRB1*10, DRB1*11,DRB1*12, DRB1*13, DRB1*14, DRB1*15, DRB1*16, DRB3*01, DRB3*02, DRB3*03,DRB4*01, and DRB5*01.
 25. The method of claim 24, wherein thesusceptible HLA allele is selected from DRB1*01:01, DRB1*01:03,DRB1*03:01, DRB1*04:01, DRB1*04:02, DRB1*04:03, DRB1*04:04, DRB1*04:05,DRB1*04:08, DRB1*07:01, DRB1*08:01, DRB1*09:01, DRB1*10:01, DRB1*11:02,DRB1*11:03, DRB1*12:01, DRB1*13:01, DRB1*14:01, DRB1*15:01, DRB1*15:02,DRB1*16:01, DRB3*01:01, DRB3*02:02, DRB3*03:01, DRB4*01:03, andDRB5*01:01.
 26. The method of claim 25, wherein the modifying thesusceptible HLA allele comprises using an RNA-guided gene editingsystem.
 27. The method of claim 25, wherein the target amino acidposition is selected from 47, 67, 70, 71, 74, 85, 86, and 71, and thesecond identity is selected from isoleucine, aspartic acid, alanine,valine, leucine, methionine, tyrosine, arginine, and phenylalanine. 28.An engineered immune cell comprising the engineered HLA allele of claim27.
 29. A protein coded for by the engineered HLA allele of claim 27.30. A mammalian expression vector comprising the engineered HLA alleleof claim 27.